HER3 antigen-binding molecules

ABSTRACT

HER3 antigen-binding molecules are disclosed. Also disclosed are nucleic acids and expression vectors encoding, compositions comprising, and methods using, the HER3 antigen-binding molecules.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/596,723, filed Oct. 8, 2019, which is a continuation of InternationalPatent Application No. PCT/EP2019/058035, filed Mar. 29, 2019, thecontents and elements of each of which are herein incorporated byreference for all purpose.

FIELD OF THE INVENTION

The present invention relates to the fields of molecular biology, morespecifically antibody technology. The present invention also relates tomethods of medical treatment and prophylaxis.

BACKGROUND TO THE INVENTION

Increased HER3 expression is linked to poor prognosis in multiple solidtumors, including breast, gastric, head & neck, pancreatic, ovarian, andlung cancers. HER3-mediated signalling has adverse consequences fortumour progression; HER3 upregulation is associated with resistance toanti-HER2 and anti-EGFR therapy, and solid tumors refractory toanti-PD-1 therapy have been shown to have higher HER3 expressioncompared to responders to anti-PD-1 therapy.

HER3-binding antibodies are described e.g. in Zhang et al., ActaBiochimica et Biophysica Sinica (2016) 48(1): 39-48. The anti-HER3antibody LJM-716 binds to an epitope on subdomains II and IV of the HER3extracellular domain, locking HER3 in the inactive conformation (Garneret al., Cancer Res (2013) 73: 6024-6035). MM-121 (also known asseribantumab) has been shown to inhibit HER3-mediated signalling byblocking binding of heregulin (HRG) to HER3 (Schoeberl et al., Sci.Signal. (2009) 2(77): ra31). Patritumab (also known as U-1287 andAMG-888) also blocks binding of heregulins to HER3 (see e.g. Shimizu etal. Cancer Chemother Pharmacol. (2017) 79(3):489-495. RG7116 (also knownas lumretuzumab and RO-5479599) recognises an epitope in subdomain I ofthe HER3 extracellular domain (see e.g. Mirschberger et al. CancerResearch (2013) 73(16) 5183-5194). KTN3379 binds to HER3 throughinteraction with amino acid residues in subdomain III (corresponding tothe following positions of SEQ ID NO:1: Gly476, Pro477, Arg481, Gly452,Arg475, Ser450, Gly420, Ala451, Gly419, Arg421, Thr394, Leu423, Arg426,Gly427, Lys356, Leu358, Leu358, Lys356, Ala330, Lys329 and Gly337), andMet310, Glu311 and Pro328 of subdomain II (see Lee et al., Proc NatlAcad Sci USA. 2015 Oct. 27; 112(43):13225). AV-203 (also known asCAN-017) has been shown to block binding of NRG1 to HER3 and to promoteHER3 degradation (see Meetze et al., Eur J Cancer 2012; 48:126).REGN1400 also inhibits binding of ligand to HER3 (see Zhang et al., MolCancer Ther (2014) 13:1345-1355). RG7597 (duligotuzumab) is a dualaction Fab (DAF) capable of binding to both HER3 and EGFR, and binds tosubdomain III of HER3 (see Schaefer et al., Cancer Cell (2011)20(4):472-486). MM-111 and MM-141 are bispecific antibodies havingHER3-binding arms which inhibit HRG ligand binding to HER3 (see McDonaghet al. Mol Cancer Ther (2012) 11:582-593 and Fitzgerald et al., MolCancer Ther (2014) 13:410-425).

SUMMARY OF THE INVENTION

In a first aspect the present invention provides an antigen-bindingmolecule, optionally isolated, which is capable of binding to HER3 inextracellular region subdomain II.

In some embodiments the antigen-binding molecule inhibits interactionbetween HER3 and an interaction partner for HER3.

In some embodiments the antigen-binding molecule is capable of bindingto a polypeptide comprising or consisting of the amino acid sequence ofSEQ ID NO:16.

In some embodiments the antigen-binding molecule is capable of bindingto a polypeptide comprising the amino acid sequence of SEQ ID NO:229.

In some embodiments the antigen-binding molecule is capable of bindingto a polypeptide comprising the amino acid sequences of SEQ ID NO:230and 231.

In some embodiments the antigen-binding molecule is capable of bindingto a polypeptide comprising the amino acid sequence of SEQ ID NO:230.

In some embodiments the antigen-binding molecule is capable of bindingto a polypeptide comprising the amino acid sequence of SEQ ID NO:231.

In some embodiments the antigen-binding molecule is capable of bindingto a polypeptide comprising the amino acid sequence of SEQ ID NO:23.

In some embodiments the antigen-binding molecule is capable of bindingto a polypeptide comprising the amino acid sequence of SEQ ID NO:21.

In some embodiments the antigen-binding molecule comprises:

-   -   (i) a heavy chain variable (VH) region incorporating the        following CDRs:        -   HC-CDR1 having the amino acid sequence of SEQ ID NO:43        -   HC-CDR2 having the amino acid sequence of SEQ ID NO:46        -   HC-CDR3 having the amino acid sequence of SEQ ID NO:51; and    -   (ii) a light chain variable (VL) region incorporating the        following CDRs:        -   LC-CDR1 having the amino acid sequence of SEQ ID NO:91        -   LC-CDR2 having the amino acid sequence of SEQ ID NO:94        -   LC-CDR3 having the amino acid sequence of SEQ ID NO:99.

In some embodiments the antigen-binding molecule comprises:

-   -   (i) a heavy chain variable (VH) region incorporating the        following CDRs:        -   HC-CDR1 having the amino acid sequence of SEQ ID NO:41        -   HC-CDR2 having the amino acid sequence of SEQ ID NO:44        -   HC-CDR3 having the amino acid sequence of SEQ ID NO:47; and    -   (ii) a light chain variable (VL) region incorporating the        following CDRs:        -   LC-CDR1 having the amino acid sequence of SEQ ID NO:88        -   LC-CDR2 having the amino acid sequence of SEQ ID NO:92        -   LC-CDR3 having the amino acid sequence of SEQ ID NO:95.

In some embodiments the antigen-binding molecule comprises:

-   -   (i) a heavy chain variable (VH) region incorporating the        following CDRs:        -   HC-CDR1 having the amino acid sequence of SEQ ID NO:41        -   HC-CDR2 having the amino acid sequence of SEQ ID NO:44        -   HC-CDR3 having the amino acid sequence of SEQ ID NO:47; and    -   (ii) a light chain variable (VL) region incorporating the        following CDRs:        -   LC-CDR1 having the amino acid sequence of SEQ ID NO:89        -   LC-CDR2 having the amino acid sequence of SEQ ID NO:92        -   LC-CDR3 having the amino acid sequence of SEQ ID NO:95.

In some embodiments the antigen-binding molecule comprises:

-   -   (i) a heavy chain variable (VH) region incorporating the        following CDRs:        -   HC-CDR1 having the amino acid sequence of SEQ ID NO:41        -   HC-CDR2 having the amino acid sequence of SEQ ID NO:44        -   HC-CDR3 having the amino acid sequence of SEQ ID NO:47; and    -   (ii) a light chain variable (VL) region incorporating the        following CDRs:        -   LC-CDR1 having the amino acid sequence of SEQ ID NO:90        -   LC-CDR2 having the amino acid sequence of SEQ ID NO:92        -   LC-CDR3 having the amino acid sequence of SEQ ID NO:96.

In some embodiments the antigen-binding molecule comprises:

-   -   (i) a heavy chain variable (VH) region incorporating the        following CDRs:        -   HC-CDR1 having the amino acid sequence of SEQ ID NO:41        -   HC-CDR2 having the amino acid sequence of SEQ ID NO:44        -   HC-CDR3 having the amino acid sequence of SEQ ID NO:47; and    -   (ii) a light chain variable (VL) region incorporating the        following CDRs:        -   LC-CDR1 having the amino acid sequence of SEQ ID NO:88        -   LC-CDR2 having the amino acid sequence of SEQ ID NO:92        -   LC-CDR3 having the amino acid sequence of SEQ ID NO:98.

In some embodiments the antigen-binding molecule comprises:

-   -   (i) a heavy chain variable (VH) region incorporating the        following CDRs:        -   HC-CDR1 having the amino acid sequence of SEQ ID NO:41        -   HC-CDR2 having the amino acid sequence of SEQ ID NO:45        -   HC-CDR3 having the amino acid sequence of SEQ ID NO:47; and    -   (ii) a light chain variable (VL) region incorporating the        following CDRs:        -   LC-CDR1 having the amino acid sequence of SEQ ID NO:88        -   LC-CDR2 having the amino acid sequence of SEQ ID NO:93        -   LC-CDR3 having the amino acid sequence of SEQ ID NO:95.

In some embodiments the antigen-binding molecule comprises:

-   -   (i) a heavy chain variable (VH) region incorporating the        following CDRs;        -   HC-CDR1 having the amino acid sequence of SEQ ID NO:41        -   HC-CDR2 having the amino acid sequence of SEQ ID NO:45        -   HC-CDR3 having the amino acid sequence of SEQ ID NO:49; and    -   (ii) a light chain variable (VL) region incorporating the        following CDRs:        -   LC-CDR1 having the amino acid sequence of SEQ ID NO:88        -   LC-CDR2 having the amino acid sequence of SEQ ID NO:93        -   LC-CDR3 having the amino acid sequence of SEQ ID NO:95.

In some embodiments the antigen-binding molecule comprises:

-   -   (i) a heavy chain variable (VH) region incorporating the        following CDRs:        -   HC-CDR1 having the amino acid sequence of SEQ ID NO:41        -   HC-CDR2 having the amino acid sequence of SEQ ID NO:45        -   HC-CDR3 having the amino acid sequence of SEQ ID NO:50; and    -   (ii) a light chain variable (VL) region incorporating the        following CDRs:        -   LC-CDR1 having the amino acid sequence of SEQ ID NO:88        -   LC-CDR2 having the amino acid sequence of SEQ ID NO:93        -   LC-CDR3 having the amino acid sequence of SEQ ID NO:95.

In some embodiments the antigen-binding molecule comprises:

-   -   (i) a heavy chain variable (VH) region incorporating the        following CDRs:        -   HC-CDR1 having the amino acid sequence of SEQ ID NO:41        -   HC-CDR2 having the amino acid sequence of SEQ ID NO:45        -   HC-CDR3 having the amino acid sequence of SEQ ID NO:48; and    -   (ii) a light chain variable (VL) region incorporating the        following CDRs:        -   LC-CDR1 having the amino acid sequence of SEQ ID NO:88        -   LC-CDR2 having the amino acid sequence of SEQ ID NO:92        -   LC-CDR3 having the amino acid sequence of SEQ ID NO:95.

In some embodiments the antigen-binding molecule comprises:

-   -   (i) a heavy chain variable (VH) region incorporating the        following CDRs:        -   HC-CDR1 having the amino acid sequence of SEQ ID NO:41        -   HC-CDR2 having the amino acid sequence of SEQ ID NO:45        -   HC-CDR3 having the amino acid sequence of SEQ ID NO:48; and    -   (ii) a light chain variable (VL) region incorporating the        following CDRs:        -   LC-CDR1 having the amino acid sequence of SEQ ID NO:88        -   LC-CDR2 having the amino acid sequence of SEQ ID NO:92        -   LC-CDR3 having the amino acid sequence of SEQ ID NO:97.

In some embodiments the antigen-binding molecule comprises;

-   -   (i) a heavy chain variable (VH) region incorporating the        following CDRs:        -   HC-CDR1 having the amino acid sequence of SEQ ID NO:42        -   HC-CDR2 having the amino acid sequence of SEQ ID NO:45        -   HC-CDR3 having the amino acid sequence of SEQ ID NO:48; and    -   (ii) a light chain variable (VL) region incorporating the        following CDRs:        -   LC-CDR1 having the amino acid sequence of SEQ ID NO:88        -   LC-CDR2 having the amino acid sequence of SEQ ID NO:92        -   LC-CDR3 having the amino acid sequence of SEQ ID NO:95.

In some embodiments the antigen-binding molecule comprises:

-   -   (i) a heavy chain variable (VH) region incorporating the        following CDRs:        -   HC-CDR1 having the amino acid sequence of SEQ ID NO:158        -   HC-CDR2 having the amino acid sequence of SEQ ID NO:159        -   HC-CDR3 having the amino acid sequence of SEQ ID NO:160; and    -   (ii) a light chain variable (VL) region incorporating the        following CDRs:        -   LC-CDR1 having the amino acid sequence of SEQ ID NO:165        -   LC-CDR2 having the amino acid sequence of SEQ ID NO:166        -   LC-CDR3 having the amino acid sequence of SEQ ID NO:167.

In some embodiments the antigen-binding molecule is capable of bindingto a polypeptide comprising the amino acid sequence of SEQ ID NO:22.

In some embodiments the antigen-binding molecule comprises:

-   -   (i) a heavy chain variable (VH) region incorporating the        following CDRs:        -   HC-CDR1 having the amino acid sequence of SEQ ID NO:128        -   HC-CDR2 having the amino acid sequence of SEQ ID NO:129        -   HC-CDR3 having the amino acid sequence of SEQ ID NO:130; and    -   (ii) a light chain variable (VL) region incorporating the        following CDRs:        -   LC-CDR1 having the amino acid sequence of SEQ ID NO:136        -   LC-CDR2 having the amino acid sequence of SEQ ID NO:137        -   LC-CDR3 having the amino acid sequence of SEQ ID NO:138.

In some embodiments the antigen-binding molecule comprises:

-   -   (i) a heavy chain variable (VH) region incorporating the        following CDRs:        -   HC-CDR1 having the amino acid sequence of SEQ ID NO:144        -   HC-CDR2 having the amino acid sequence of SEQ ID NO:145        -   HC-CDR3 having the amino acid sequence of SEQ ID NO:146; and    -   (ii) a light chain variable (VL) region incorporating the        following CDRs:        -   LC-CDR1 having the amino acid sequence of SEQ ID NO:151        -   LC-CDR2 having the amino acid sequence of SEQ ID NO:152        -   LC-CDR3 having the amino acid sequence of SEQ ID NO:153.

In particular embodiments the antigen-binding molecule comprises:

-   -   (i) a heavy chain variable (VH) region incorporating the        following CDRs:        -   HC-CDR1 having the amino acid sequence of SEQ ID NO:41        -   HC-CDR2 having the amino acid sequence of SEQ ID NO:45        -   HC-CDR3 having the amino acid sequence of SEQ ID NO:48; and    -   (ii) a light chain variable (VL) region incorporating the        following CDRs:        -   LC-CDR1 having the amino acid sequence of SEQ ID NO:88        -   LC-CDR2 having the amino acid sequence of SEQ ID NO:92        -   LC-CDR3 having the amino acid sequence of SEQ ID NO:95;            or    -   (i) a heavy chain variable (VH) region incorporating the        following CDRs:        -   HC-CDR1 having the amino acid sequence of SEQ ID NO:41        -   HC-CDR2 having the amino acid sequence of SEQ ID NO:45        -   HC-CDR3 having the amino acid sequence of SEQ ID NO:48; and    -   (ii) a light chain variable (VL) region incorporating the        following CDRs:        -   LC-CDR1 having the amino acid sequence of SEQ ID NO:88        -   LC-CDR2 having the amino acid sequence of SEQ ID NO:92        -   LC-CDR3 having the amino acid sequence of SEQ ID NO:97;            or    -   (i) a heavy chain variable (VH) region incorporating the        following CDRs:        -   HC-CDR1 having the amino acid sequence of SEQ ID NO:42        -   HC-CDR2 having the amino acid sequence of SEQ ID NO:45        -   HC-CDR3 having the amino acid sequence of SEQ ID NO:48; and    -   (ii) a light chain variable (VL) region incorporating the        following CDRs:        -   LC-CDR1 having the amino acid sequence of SEQ ID NO:88        -   LC-CDR2 having the amino acid sequence of SEQ ID NO:92        -   LC-CDR3 having the amino acid sequence of SEQ ID NO:95.

In some embodiments the antigen-binding molecule comprises:

-   -   (i) a VH region comprising an amino acid sequence having at        least 70% sequence identity to the amino acid sequence of SEQ ID        NO:24; and        -   a VL region comprising an amino acid sequence having at            least 70% sequence identity to the amino acid sequence of            SEQ ID NO:74;            or    -   (ii) a VH region comprising an amino acid sequence having at        least 70% sequence identity to the amino acid sequence of SEQ ID        NO:25; and        -   a VL region comprising an amino acid sequence having at            least 70% sequence identity to the amino acid sequence of            SEQ ID NO:75;            or    -   (iii) a VH region comprising an amino acid sequence having at        least 70% sequence identity to the amino acid sequence of SEQ ID        NO:26; and        -   a VL region comprising an amino acid sequence having at            least 70% sequence identity to the amino acid sequence of            SEQ ID NO:78;            or    -   (iv) a VH region comprising an amino acid sequence having at        least 70% sequence identity to the amino acid sequence of SEQ ID        NO:27; and        -   a VL region comprising an amino acid sequence having at            least 70% sequence identity to the amino acid sequence of            SEQ ID NO:77;            or    -   (v) a VH region comprising an amino acid sequence having at        least 70% sequence identity to the amino acid sequence of SEQ ID        NO:28; and        -   a VL region comprising an amino acid sequence having at            least 70% sequence identity to the amino acid sequence of            SEQ ID NO:78;            or    -   (vi) a VH region comprising an amino acid sequence having at        least 70% sequence identity to the amino acid sequence of SEQ ID        NO:29; and        -   a VL region comprising an amino acid sequence having at            least 70% sequence identity to the amino acid sequence of            SEQ ID NO:78;            or    -   (vii) a VH region comprising an amino acid sequence having at        least 70% sequence identity to the amino acid sequence of SEQ ID        NO:30; and        -   a VL region comprising an amino acid sequence having at            least 70% sequence identity to the amino acid sequence of            SEQ ID NO:78;            or    -   (viii) a VH region comprising an amino acid sequence having at        least 70% sequence identity to the amino acid sequence of SEQ ID        NO:31; and        -   a VL region comprising an amino acid sequence having at            least 70% sequence identity to the amino acid sequence of            SEQ ID NO:79;            or    -   (ix) a VH region comprising an amino acid sequence having at        least 70% sequence identity to the amino acid sequence of SEQ ID        NO:32; and        -   a VL region comprising an amino acid sequence having at            least 70% sequence identity to the amino acid sequence of            SEQ ID NO:79;            or    -   (x) a VH region comprising an amino acid sequence having at        least 70% sequence identity to the amino acid sequence of SEQ ID        NO:33; and        -   a VL region comprising an amino acid sequence having at            least 70% sequence identity to the amino acid sequence of            SEQ ID NO:80;            or    -   (xi) a VH region comprising an amino acid sequence having at        least 70% sequence identity to the amino acid sequence of SEQ ID        NO:34; and        -   a VL region comprising an amino acid sequence having at            least 70% sequence identity to the amino acid sequence of            SEQ ID NO:81;            or    -   (xii) a VH region comprising an amino acid sequence having at        least 70% sequence identity to the amino acid sequence of SEQ ID        NO:35; and        -   a VL region comprising an amino acid sequence having at            least 70% sequence identity to the amino acid sequence of            SEQ ID NO:82;            or    -   (xiii) a VH region comprising an amino acid sequence having at        least 70% sequence identity to the amino acid sequence of SEQ ID        NO:36; and        -   a VL region comprising an amino acid sequence having at            least 70% sequence identity to the amino acid sequence of            SEQ ID NO:83;            or    -   (xiv) a VH region comprising an amino acid sequence having at        least 70% sequence identity to the amino acid sequence of SEQ ID        NO:37; and        -   a VL region comprising an amino acid sequence having at            least 70% sequence identity to the amino acid sequence of            SEQ ID NO:84;            or    -   (xv) a VH region comprising an amino acid sequence having at        least 70% sequence identity to the amino acid sequence of SEQ ID        NO:38; and        -   a VL region comprising an amino acid sequence having at            least 70% sequence identity to the amino acid sequence of            SEQ ID NO:85;            or    -   (xvi) a VH region comprising an amino acid sequence having at        least 70% sequence identity to the amino acid sequence of SEQ ID        NO:39; and        -   a VL region comprising an amino acid sequence having at            least 70% sequence identity to the amino acid sequence of            SEQ ID NO:86;            or    -   (xvii) a VH region comprising an amino acid sequence having at        least 70% sequence identity to the amino acid sequence of SEQ ID        NO:40; and        -   a VL region comprising an amino acid sequence having at            least 70% sequence identity to the amino acid sequence of            SEQ ID NO:87;            or    -   (xviii) a VH region comprising an amino acid sequence having at        least 70% sequence identity to the amino acid sequence of SEQ ID        NO:127; and        -   a VL region comprising an amino acid sequence having at            least 70% sequence identity to the amino acid sequence of            SEQ ID NO:135:            or    -   (xix) a VH region comprising an amino acid sequence having at        least 70% sequence identity to the amino acid sequence of SEQ ID        NO:143; and        -   a VL region comprising an amino acid sequence having at            least 70% sequence identity to the amino acid sequence of            SEQ ID NO:150;            or    -   (xx) a VH region comprising an amino acid sequence having at        least 70% sequence identity to the amino acid sequence of SEQ ID        NO:157; and        -   a VL region comprising an amino acid sequence having at            least 70% sequence identity to the amino acid sequence of            SEQ ID NO:164.

In particular embodiments the antigen-binding molecule comprises:

-   -   (i) a VH region comprising an amino acid sequence having at        least 70% sequence identity to the amino acid sequence of SEQ ID        NO:36; and        -   a VL region comprising an amino acid sequence having at            least 70% sequence identity to the amino acid sequence of            SEQ ID NO:83;            or    -   (ii) a VH region comprising an amino acid sequence having at        least 70% sequence identity to the amino acid sequence of SEQ ID        NO:37; and        -   a VL region comprising an amino acid sequence having at            least 70% sequence identity to the amino acid sequence of            SEQ ID NO:84;            or    -   (iii) a VH region comprising an amino acid sequence having at        least 70% sequence identity to the amino acid sequence of SEQ ID        NO:38; and        -   a VL region comprising an amino acid sequence having at            least 70% sequence identity to the amino acid sequence of            SEQ ID NO:85.

In some embodiments the antigen-binding molecule is capable of bindingto human HER3 and one or more of mouse HER3, rat HER3 and cynomolgousmacaque HER3.

Also provided is an antigen-binding molecule, optionally isolated,comprising (i) an antigen-binding molecule according to the invention,and (ii) an antigen-binding molecule capable of binding to an antigenother than HER3.

In some embodiments the antigen-binding molecule is capable of bindingto cells expressing HER3 at the cell surface.

In some embodiments the antigen-binding molecule is capable ofinhibiting HER3-mediated signalling.

In some embodiments the antigen-binding molecule comprises an Fc region,the Fc region comprising a polypeptide having: (i) C at the positioncorresponding to position 242, and C at the position corresponding toposition 334, and (ii) one or more of: A at the position correspondingto position 236, D at the position corresponding to position 239, E atthe position corresponding to position 332, L at the positioncorresponding to position 330, K at the position corresponding toposition 345, and G at the position corresponding to position 430. Insome embodiments the Fc region comprises a polypeptide having C at theposition corresponding to position 242, C at the position correspondingto position 334, A at the position corresponding to position 236, D atthe position corresponding to position 239, E at the positioncorresponding to position 332, and L at the position corresponding toposition 330.

Also provided is a chimeric antigen receptor (CAR) comprising anantigen-binding molecule according to the present invention.

Also provided is a nucleic acid, or a plurality of nucleic acids,optionally isolated, encoding an antigen-binding molecule or a CARaccording to the present invention.

Also provided is an expression vector, or a plurality of expressionvectors, comprising a nucleic acid or a plurality of nucleic acidsaccording to the present invention.

Also provided is a cell comprising an antigen-binding molecule, a CAR, anucleic acid or a plurality of nucleic acids, or an expression vector ora plurality of expression vectors according to the present invention.

Also provided is a method comprising culturing a cell comprising anucleic acid or a plurality of nucleic acids or an expression vector ora plurality of expression vectors according to the present invention,under conditions suitable for expression of the antigen-binding moleculeor CAR from the nucleic acid(s) or expression vector(s).

Also provided is a composition comprising an antigen-binding molecule, aCAR, a nucleic acid or a plurality of nucleic acids, an expressionvector or a plurality of expression vectors, or a cell according to thepresent invention.

Also provided is an antigen-binding molecule, a CAR, a nucleic acid or aplurality of nucleic acids, an expression vector or a plurality ofexpression vectors, a cell, or a composition according to the presentinvention for use in a method of medical treatment or prophylaxis.

Also provided is an antigen-binding molecule, a CAR, a nucleic acid or aplurality of nucleic acids, an expression vector or a plurality ofexpression vectors, a cell, or a composition according to the presentinvention for use in a method of treatment or prevention of a cancer.

Also provided is the use of an antigen-binding molecule, a CAR, anucleic acid or a plurality of nucleic acids, an expression vector or aplurality of expression vectors, a cell, or a composition according tothe present invention in the manufacture of a medicament for use in amethod of treatment or prevention of a cancer.

Also provided is a method of treating or preventing a cancer, comprisingadministering to a subject a therapeutically or prophylacticallyeffective amount of an antigen-binding molecule, a CAR, a nucleic acidor a plurality of nucleic acids, an expression vector or a plurality ofexpression vectors, a cell, or a composition according to the presentinvention.

In some embodiments in accordance with various aspects of the presentinvention, the method additionally comprises administration of aninhibitor of signalling mediated by an EGFR family member, optionallywherein the inhibitor of signalling mediated by an EGFR family member isan inhibitor of signalling mediated by HER2 and/or EGFR.

In some embodiments the cancer is selected from: a cancer comprisingcells expressing an EGFR family member, a cancer comprising cellsexpressing HER3, a solid tumor, breast cancer, breast carcinoma, ductalcarcinoma, gastric cancer, gastric carcinoma, gastric adenocarcinoma,colorectal cancer, colorectal carcinoma, colorectal adenocarcinoma, headand neck cancer, squamous cell carcinoma of the head and neck (SCCHN),lung cancer, lung adenocarcinoma, squamous cell lung carcinoma, ovariancancer, ovarian carcinoma, ovarian serous adenocarcinoma, kidney cancer,renal cell carcinoma, renal clear cell carcinoma, renal celladenocarcinoma, renal papillary cell carcinoma, pancreatic cancer,pancreatic adenocarcinoma, pancreatic ductal adenocarcinoma, cervicalcancer, cervical squamous cell carcinoma, skin cancer, melanoma,esophageal cancer, esophageal adenocarcinoma, liver cancer,hepatocellular carcinoma, cholangiocarcinoma, uterine cancer, uterinecorpus endometrial carcinoma, thyroid cancer, thyroid carcinoma,pheochromocytoma, paraganglioma, bladder cancer, bladder urothelialcarcinoma, prostate cancer, prostate adenocarcinoma, sarcoma andthymoma.

Also provided is a method of inhibiting HER3-mediated signalling,comprising contacting HER3-expressing cells with an antigen-bindingmolecule according to the present invention.

Also provided is a method of reducing the number or activity ofHER3-expressing cells, the method comprising contacting HER3-expressingcells with an antigen-binding molecule according to the presentinvention.

Also provided is an in vitro complex, optionally isolated, comprising anantigen-binding molecule according to the present invention bound toHER3.

Also provided is a method comprising contacting a sample containing, orsuspected to contain, HER3 with an antigen-binding molecule according tothe present invention, and detecting the formation of a complex of theantigen-binding molecule with HER3.

Also provided is a method of selecting or stratifying a subject fortreatment with a HER3-targeted agent, the method comprising contacting,in vitro, a sample from the subject with an antigen-binding moleculeaccording to the present invention and detecting the formation of acomplex of the antigen-binding molecule with HER3.

Also provided is the use of an antigen-binding molecule according to thepresent invention as an in vitro or in vivo diagnostic or prognosticagent.

Also provided is the use of an antigen-binding molecule according to thepresent invention in a method for detecting, localizing or imaging acancer, optionally wherein the cancer is selected from: a cancercomprising cells expressing an EGFR family member, a cancer comprisingcells expressing HER3, a sold tumor, breast cancer, breast carcinoma,ductal carcinoma, gastric cancer, gastric carcinoma, gastricadenocarcinoma, colorectal cancer, colorectal carcinoma, colorectaladenocarcinoma, head and neck cancer, squamous cell carcinoma of thehead and neck (SCCHN), lung cancer, lung adenocarcinoma, squamous celllung carcinoma, ovarian cancer, ovarian carcinoma, ovarian serousadenocarcinoma, kidney cancer, renal cell carcinoma, renal clear cellcarcinoma, renal cell adenocarcinoma, renal papillary cell carcinoma,pancreatic cancer, pancreatic adenocarcinoma, pancreatic ductaladenocarcinoma, cervical cancer, cervical squamous cell carcinoma, skincancer, melanoma, esophageal cancer, esophageal adenocarcinoma, livercancer, hepatocellular carcinoma, cholangiocarcinoma, uterine cancer,uterine corpus endometrial carcinoma, thyroid cancer, thyroid carcinoma,pheochromocytoma, paraganglioma, bladder cancer, bladder urothelialcarcinoma, prostate cancer, prostate adenocarcinoma, sarcoma andthymoma.

DESCRIPTION

The present invention relates to novel HER3-binding molecules havingimproved properties as compared to known anti-HER3 antibodies.

The inventors undertook the targeted generation of antigen-bindingmolecules which bind to particular regions of interest in theextracellular region of HER3. The HER3-binding molecules of the presentinvention are provided with combinations of desirable biophysical and/orfunctional properties as compared to antigen-binding molecules disclosedin the prior art.

In embodiments of the present invention the antigen binding moleculesare capable of binding to the subdomain II of the extracellular regionof HER3 (SEQ ID NO:16), and inhibit association of the bound HER3molecule with interaction partners.

In particular, HER3-binding antigen-binding molecules described hereinare demonstrated to bind to an epitope of HER3 providing for potentinhibition of association of HER3 with interaction partners, stronginhibition of downstream signalling and exceptional anti-cancer activityagainst a wide range of cancers.

HER3

HER3 (also known e.g. as ERBB3 LCCS2, MDA-BF-1) is the proteinidentified by UniProt P21860. Alternative splicing of mRNA encoded bythe human ERBB3 gene yields five different isoforms: isoform 1 (UniProt;P21860-1, v1; SEQ ID NO:1); isoform 2 (UniProt: P21860-2, SEQ ID NO:2),which comprises a different sequence to SEQ ID NO:1 from position 141,and which lacks amino acid sequence corresponding to positions 183 to1342 of SEQ ID NO:1; isoform 3 (UniProt: P21860-3; SEQ ID NO:3), whichcomprises the substitution C331F relative to SEQ ID NO:1, and whichlacks the amino acid sequence corresponding to positions 332 to 1342 ofSEQ ID NO:1; isoform 4 (UniProt: P21860-4; SEQ 10 NO:4), which lacks theamino acid sequence corresponding to positions 1 to 59 of SEQ ID NO:1;and isoform 5 (UniProt: P21360-5; SEQ ID NO:5), which lacks the aminoacid sequence corresponding to positions 1 to 643 of SEQ ID NO:1.

The N-terminal 19 amino acids of SEQ ID NOs:1 to 3 constitute a signalpeptide, and so the mature form of HER3 isoforms 1, 2 and 3 (i.e. afterprocessing to remove the signal peptide) have the amino acid sequencesshown in SEQ ID NOs:6, 7 and 8, respectively.

The structure and function of HER3 is described e.g. in Cho and LeahyScience (2002) 297 (5585):1330-1333, Singer et al., Journal ofBiological Chemistry (2001) 276, 44266-44274, Roskoski et al.,Pharmacol. Res. (2014) 79: 34-74, Bazley and Gullick Endocrine-RelatedCancer (2005) S17-S27 and Mujoo et al., Oncotarget (2014)5(21):10222-10236, each of which are hereby incorporated by reference intheir entirety. HER3 is a single-pass transmembrane ErbB receptortyrosine kinase having an N-terminal extracellular region (SEQ ID NO:9)comprising two leucine-rich subdomains (domains I and III, shown in SEQID NOs:15 and 17, respectively) and two cysteine-rich subdomains(domains II and IV, shown in SEQ ID NOs:16 and 18, respectively). DomainII comprises a β hairpin dimerisation loop (SEQ ID NO:19) which isinvolved in intermolecular interaction with other HER receptormolecules. The extracellular region is linked via a transmembrane region(SEQ ID NO:10) to a cytoplasmic region (SEQ ID NO:11). The cytoplasmicregion comprises a juxtamembrane segment (SEQ ID NO:12), a proteinkinase domain (SEQ ID NO:13), and a C-terminal segment (SEQ ID NO:14).

Signalling through HER3 involves receptor homodimerisation (i.e. withother HER3 receptors) or heterodimerisation (with other HER receptors,e.g. HER2) and consequent autophosphotylation by the protein kinasedomain of tyrosines of the cytoplasmic region. The phosphotylatedtyrosine residues recruit adaptor/effector proteins (e.g. Grb2 andphospholipase Cγ (PLCγ), containing src homology domain 2 (SH2) orphosphotyrosine binding (PTB) domains.

Signalling through HER3 can be activated in a ligand-dependent orligand-independent manner. In the absence of ligand, HER3 receptormolecules are normally expressed at the cell surface as monomers with aconformation which prevents receptor dimerisation in which thedimerisation loop of subdomain II makes intramolecular contact with apocket on subdomain IV. Binding of a HER3 ligand such as a neuregulin(NRG), e.g. NRG1 (also known as heregulin, HRG) or NRG2 to subdomains Iand III of the extracellular region causes a conformational change whichresults in the exposure of the dimerisation loop of subdomain II,facilitating receptor dimerisation and signalling. Somecancer-associated mutations in HER3 may disrupt interaction ofsubdomains II and IV required for the formation of the inactive ‘closed’conformation and thereby cause constitutive presentation of thedimerisation loop and activation of HER3-mediated signalling in theabsence of ligand binding (see e.g. in Jaiswal et al., Cancer Cell(2013) 23(5): 603-617).

In this specification “HER3” refers to HER3 from any species andincludes HER3 isoforms, fragments, variants (including mutants) orhomologues from any species.

As used herein, a “fragment”, “variant” or “homologue” of a protein mayoptionally be characterised as having at least 60%, preferably one of70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or100% amino acid sequence identity to the amino acid sequence of thereference protein (e.g. a reference isoform). In some embodimentsfragments, variants, isoforms and homologues of a reference protein maybe characterised by ability to perform a function performed by thereference protein.

A “fragment” generally refers to a fraction of the reference protein. A“variant” generally refers to a protein having an amino acid sequencecomprising one or more amino acid substitutions, insertions, deletionsor other modifications relative to the amino acid sequence of thereference protein, but retaining a considerable degree of sequenceidentity (e.g. at least 60%) to the amino acid sequence of the referenceprotein. An “isoform” generally refers to a variant of the referenceprotein expressed by the same species as the species of the referenceprotein (e.g. HER3 isoforms 1 to 5 are all isoforms of one another). A“homologue” generally refers to a variant of the reference proteinproduced by a different species as compared to the species of thereference protein. For example, human HER3 isoform 1 (P21860-1, v1; SEQID NO:1) and Rhesus macaque HER3 (UniProt; F7HEH3-1, v2; SEQ ID NO:20)are homologues of one another. Homologues include orthologues.

A “fragment” of a reference protein may be of any length (by number ofamino acids), although may optionally be at least 20% of the length ofthe reference protein (that is, the protein from which the fragment isderived) and may have a maximum length of one of 50%, 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the length of thereference protein.

A fragment of HER3 may have a minimum length of one of 10, 20, 30, 40,50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900,1000, 1100, 1200 amino acids, and may have a maximum length of one of20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700,800, 900, 1000, 1100, 1200, or 1300 amino acids.

In some embodiments, the HER3 is HER3 from a mammal (e.g. a primate(rhesus, cynomolgous, non-human primate or human) and/or a rodent (e.g.rat or murine) HER3). Isoforms, fragments, variants or homologues ofHER3 may optionally be characterised as having at least 70%, preferablyone of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or100% amino acid sequence identity to the amino acid sequence of animmature or mature HER3 isoform from a given species, e.g. human.

Isoforms, fragments, variants or homologues may optionally be functionalisoforms, fragments, variants or homologues, e.g. having a functionalproperty/activity of the reference HER3 (e.g. human HER3 isoform 1), asdetermined by analysis by a suitable assay for the functionalproperty/activity. For example, an isoform, fragment, variant orhomologue of HER3 may display association with one or more of: HER2,NRG1 (type I, II, III, IV, V or VI) or NRG2 (α or β).

In some embodiments, the HER3 comprises, or consists of, an amino acidsequence having at least 70%, preferably one of 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identityto one of SEQ ID NOs:1 to 8.

In some embodiments, a fragment of HER3 comprises, or consists of, anamino acid sequence having at least 70%, preferably one of 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acidsequence identity to one of SEQ ID NOs:9 to 19, e.g. one of 9, 16 or 19.

Regions of Particular Interest on the Target Molecule

The antigen-binding molecules of the present invention were specificallydesigned to target regions of HER3 of particular interest. In a two-stepapproach, HER3 regions to be targeted were selected following analysisfor predicted antigenicity, function and safety. Antibodies specific forthe target regions of HER3 were then prepared using peptidescorresponding to the target regions as immunogens to raise specificmonoclonal antibodies, and subsequent screening identified antibodiescapable of binding to HER3 in the native state. This approach providesexquisite control over the antibody epitope.

The antigen-binding molecules of the present invention may be defined byreference to the region of HER3 to which they bind. The antigen-bindingmolecules of the present invention may bind to a particular region ofinterest of HER3. In some embodiments the antigen-binding molecule maybind to a linear epitope of HER3, consisting of a contiguous sequence ofamino acids (i.e. an amino acid primary sequence). In some embodiments,the antigen-binding molecule may bind to a conformational epitope ofHER3, consisting of a discontinuous sequence of amino acids of the aminoacid sequence.

In some embodiments, the antigen-binding molecule of the presentinvention binds to HER3. In some embodiments, the antigen-bindingmolecule binds to the extracellular region of HER3 (e.g. the regionshown in SEQ ID NO:9). In some embodiments, the antigen-binding moleculebinds to subdomain II of the extracellular region of HER3 (e.g. theregion shown in SEQ ID NO:16).

In some embodiments, the antigen-binding molecule binds to the region ofHER3 shown in SEQ ID NO:229. In some embodiments the antigen-bindingmolecule contacts one or more amino acid residues of the region of HER3shown in SEQ ID NO:229. In some embodiments, the antigen-bindingmolecule binds to the regions of HER3 shown in SEQ ID NOs:230 and 231.In some embodiments the antigen-binding molecule contacts one or moreamino acid residues of the regions of HER3 shown in SEQ ID NOs:230 and231. In some embodiments, the antigen-binding molecule binds to theregion of HER3 shown in SEQ ID NO:230. In some embodiments theantigen-binding molecule contacts one or more amino acid residues of theregion of HER3 shown in SEQ ID NO:230. In some embodiments, theantigen-binding molecule binds to the region of HER3 shown in SEQ IDNO:231. In some embodiments the antigen-binding molecule contacts one ormore amino acid residues of the region of HER3 shown in SEQ ID NO:231.

In some embodiments, the antigen-binding molecule binds to the region ofHER3 shown in SEQ ID NO:23. In some embodiments the antigen-bindingmolecule contacts one or more amino acid residues of the region of HER3shown in SEQ ID NO:23. In some embodiments, the antigen-binding moleculebinds to the region of HER3 shown in SEQ ID NO:21. In some embodimentsthe antigen-binding molecule contacts one or more amino acid residues ofthe region of HER3 shown in SEQ ID NO:21. In some embodiments theantigen-binding molecule binds to the region of HER3 shown in SEQ IDNO:19. In some embodiments the antigen-binding molecule contacts one ormore amino acid residues of the region of HER3 shown in SEQ ID NO:19. Insome embodiments, the antigen-binding molecule binds to the region ofHER3 shown in SEQ ID NO:22. In some embodiments the antigen-bindingmolecule contacts one or more amino acid residues of the region of HER3shown in SEQ ID NO:22.

In some embodiments, the antigen-binding molecule does not bind to theregion of HER3 corresponding to positions 260 to 279 of SEQ ID NO:1. Insome embodiments the antigen-binding molecule does not contact an aminoacid residue of the region of HER3 corresponding to positions 260 to 279of SEQ ID NO:1. In some embodiments, the antigen-binding molecule doesnot bind to the region of HER3 shown in SEQ ID NO:23. In someembodiments the antigen-binding molecule does not contact an amino acidresidue of the region of HER3 shown in SEQ ID NO:23.

The region of a peptide/polypeptide to which an antibody binds can bedetermined by the skilled person using various methods well known in theart, including X-ray co-crystallography analysis of antibody-antigencomplexes, peptide scanning, mutagenesis mapping, hydrogen-deuteriumexchange analysis by mass spectrometry, phage display, competition ELISAand proteolysis-based ‘protection’ methods. Such methods are described,for example, in Gershoni et al., BioDrugs, 2007, 21(3):145-156, which ishereby incorporated by reference in its entirety.

In some embodiments the antigen-binding molecule is capable of bindingthe same region of HER3, or an overlapping region of HER3, to the regionof HER3 which is bound by an antibody comprising the VH and VL sequencesof one of antibody clones 10D1, 10D1_c75, 10D1_c76, 10D1_c77,10D1_c78v1, 10D1_c78v2, 10D1_11B, 10D1_c85v1, 10D1_c85v2, 10D1_c85o1,10D1_c85o2, 10D1_c87, 10D1_c89, 10D1_c90, 10D1_c91, 10D1_c92, 10D1_c93,10A6, 4-35-B2 or 4-35-84 described herein. In some embodiments theantigen-binding molecule is capable of binding the same region of HER3,or an overlapping region of HER3, to the region of HER3 which is boundby an antibody comprising the VH and VL sequences of one of antibodyclones 10D1_c89, 10D1_c90 or 10D1_c91. In some embodiments theantigen-binding molecule is capable of binding the same region of HER3,or an overlapping region of HER3, to the region of HER3 which is boundby an antibody comprising the VH and VL sequences of antibody clone10D1_c89.

As used herein, a “peptide” refers to a chain of two or more amino acidmonomers linked by peptide bonds. A peptide typically has a length inthe region of about 2 to 50 amino acids. “polypeptide” is a polymerchain of two or more peptides. Polypeptides typically have a lengthgreater than about 50 amino acids.

In some embodiments, the antigen-binding molecule of the presentinvention is capable of binding to a polypeptide comprising, orconsisting of, the amino acid sequence of one of SEQ ID NOs:1, 3, 4, 6or 8.

In some embodiments, the antigen-binding molecule is capable of bindingto a polypeptide comprising, or consisting of, the amino acid sequenceof SEQ ID NO:9. In some embodiments, the antigen-binding molecule iscapable of binding to a polypeptide comprising, or consisting of, theamino acid sequence of SEQ ID NO:16.

In some embodiments, the antigen-binding molecule is capable of bindingto a peptide/polypeptide comprising, or consisting of, the amino acidsequence of SEQ ID NO:229. In some embodiments, the antigen-bindingmolecule is capable of binding to a peptide/polypeptide comprising, orconsisting of, the amino acid sequences of SEQ ID NOs:230 and 231. Insome embodiments, the antigen-binding molecule is capable of binding toa peptide/polypeptide comprising, or consisting of, the amino acidsequence of SEQ ID NO:230. In some embodiments, the antigen-bindingmolecule is capable of binding to a peptide; polypeptide comprising, orconsisting of, the amino acid sequence of SEQ ID NO:231. In someembodiments, the antigen-binding molecule is capable of binding to apeptide/polypeptide comprising, or consisting of, the amino acidsequence of SEQ ID NO:23. In some embodiments, the antigen-bindingmolecule is capable of binding to a peptide/polypeptide comprising, orconsisting of, the amino acid sequence of SEQ ID NO:21. In someembodiments, the antigen-binding molecule is capable of binding to apeptide/polypeptide comprising, or consisting of, the amino acidsequence of SEQ ID NO:19. In some embodiments, the antigen-bindingmolecule is capable of binding to a peptide/polypeptide comprising, orconsisting of, the amino acid sequence of SEQ ID NO:22.

In some embodiments, the antigen-binding molecule is not capable ofbinding to a peptide consisting of the amino acid sequence correspondingto positions 260 to 279 of SEQ ID NO:1. In some embodiments, theantigen-binding molecule is not capable of binding to a peptideconsisting of the amino acid sequence of SEQ 110 NO:23.

The ability of an antigen-binding molecule to bind to a givenpeptide/polypeptide can be analysed by methods well known to the skilledperson, including analysis by ELISA, immunoblot (e.g. western blot),immunoprecipitation, Surface Plasmon Resonance (SPR; see e.g. Hearty etal., Methods Mol Biol (2012) 907:411-442) or Bio-Layer Interferometry(see e.g. Lad et al., (2015) J Biomol Screen 20(4): 498-507).

In embodiments where the antigen binding molecule is capable of bindingto a peptide/polypeptide comprising a reference amino acid sequence, thepeptide/polypeptide may comprise one or more additional amino acids atone or both ends of the reference amino acid sequence. In someembodiments the peptide/polypeptide comprises e.g. 1-5, 1-10, 1-20,1-30, 1-40, 1-50, 5-10, 5-20, 5-30, 5-40, 5-50, 10-20, 10-30, 10-40,10-50, 20-30, 20-40 or 20-50 additional amino acids at one or both endsof the reference amino acid sequence.

In some embodiments the additional amino acid(s) provided at one or bothends (i.e. the N-terminal and C-terminal ends) of the reference sequencecorrespond to the positions at the ends of the reference sequence in thecontext of the amino acid sequence of HER3. By way of example, where theantigen-binding molecule is capable of binding to a peptide comprisingthe sequence of SEQ ID NO:23 and an additional two amino acids at theC-terminal end of SEQ ID NO:23, the additional two amino acids may bethreonine and lysine, corresponding to positions 278 and 279 of SEQ IDNO:1.

In some embodiments the antigen-binding molecule is capable of bindingto a peptide/polypeptide which is bound by an antibody comprising the VHand VL sequences of one of antibody clones 10D1, 10D1_c75, 10D1_c76,10D1_c77, 10D1_c78v1, 10D1_c78v2, 10D1_11B, 10D1_c85v1, 10D1_c85v2,10D1_c85o1, 10D1_c85o2, 10D1_c87, 10D1_c89, 10D1_c90, 10D1_c91,10D1_c92, 10D1_c93, 10A6, 4-35-B2 or 4-35-B4 described herein. In someembodiments the antigen-binding molecule is capable of binding to apeptide/polypeptide which is bound by an antibody comprising the VH andVL sequences of one of antibody clones 10D1_c89, 10D1_c90 or 10D1_c91.In some embodiments the antigen-binding molecule is capable of bindingto a peptide/polypeptide which is bound by an antibody comprising the VHand VL sequences of antibody clone 10D1_c89.

Antigen-Binding Molecules

The present invention provides antigen-binding molecules capable ofbinding to HER3.

An “antigen-binding molecule” refers to a molecule which is capable ofbinding to a target antigen, and encompasses monoclonal antibodies,polyclonal antibodies, monospecific and multispecific antibodies (e.g.,bispecific antibodies), and antibody fragments (e.g. Fv, scFv, Fab,scFab, F(ab′)₂, Fab₂, diabodies, triabodies, scFv-Fc, minibodies, singledomain antibodies (e.g. VhH), etc.), as long as they display binding tothe relevant target molecule(s).

The antigen-binding molecule of the present invention comprises a moietycapable of binding to a target antigen(s). In some embodiments, themoiety capable of binding to a target antigen comprises an antibodyheavy chain variable region (VH) and an antibody light chain variableregion (VL) of an antibody capable of specific binding to the targetantigen. In some embodiments, the moiety capable of binding to a targetantigen comprises or consists of an aptamer capable of binding to thetarget antigen, e.g, a nucleic acid aptamer (reviewed, for example, inZhou and Rossi Nat Rev Drug Discov, 2017 16(3):181-202). In someembodiments, the moiety capable of binding to a target antigen comprisesor consists of a antigen-binding peptide/polypeptide, e.g. a peptideaptamer, thioredoxin, monobody, anticalin, Kunitz domain, avimer,knottin, fynomer, atrimer, DARPin, affibody, nanobody (i.e. asingle-domain antibody (sdAb)) affilin, armadillo repeat protein(ArmRP), OBody or fibronectin—reviewed e.g. in Reverdatto et al., CurrTop Med Chem. 2015; 15(12): 1082-1101, which is hereby incorporated byreference in its entirety (see also e.g. Boersma et al., J Biol Chem(2011) 286:41273-85 and Emanuel et al., Mabs (2011) 3:38-48).

The antigen-binding molecules of the present invention generallycomprise an antigen-binding domain comprising a VH and a VL of anantibody capable of specific binding to the target antigen. Theantigen-binding domain formed by a VH and a VL may also be referred toherein as an Fv region.

An antigen-binding molecule may be, or may comprise, an antigen-bindingpolypeptide, or an antigen-binding polypeptide complex. Anantigen-binding molecule may comprise more than one polypeptide whichtogether form an antigen-binding domain. The polypeptides may associatecovalently or non-covalently. In some embodiments the polypeptides formpart of a larger polypeptide comprising the polypeptides (e.g. in thecase of scFv comprising VH and VL, or in the case of scFab comprisingVH-CH1 and VL-CL).

An antigen-binding molecule may refer to a non-covalent or covalentcomplex of more than one polypeptide (e.g. 2, 3, 4, 6, or 8polypeptides), e.g. an IgG-like antigen-binding molecule comprising twoheavy chain polypeptides and two light chain polypeptides.

The antigen-binding molecules of the present invention may be designedand prepared using the sequences of monoclonal antibodies (mAbs) capableof binding to HER3. Antigen-binding regions of antibodies, such assingle chain variable fragment (scFv), Fab and F(ab′)₂ fragments mayalso be used/provided. An “antigen-binding region” is any fragment of anantibody which is capable of binding to the target for which the givenantibody is specific.

Antibodies generally comprise six complementarity-determining regionsCDRs; three in the heavy chain variable (VH) region: HC-CDR1, HC-CDR2and HC-CDR3, and three in the light chain variable (VL) region: LC-CDR1,LC-CDR2, and LC-CDR3. The six CDRs together define the paratope of theantibody, which is the part of the antibody which binds to the targetantigen.

The VH region and VL region comprise framework regions (FRs) either sideof each CDR, which provide a scaffold for the CDRs. From N-terminus toC-terminus, VH regions comprise the following structure: Nterm-[HC-FR1]-[HC-CDR1]-[HC-FR2]-[HC-CDR2]-[HC-FR3]-[HC-CDR3]-[HC-FR4]-Cterm; and VL regions comprise the following structure: Nterm-[LC-FR1]-[LC-CDR1]-[LCFR2]-[LC-CDR2]-[LC-FR3]-[LC-CDR3]-[LC-FR4]-Cterm.

There are several different conventions for defining antibody OCRs andFRs, such as those described in Rabat et al., Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991), Chothia et al., J. Mol.Biol. 196:901-917 (1987), and VBASE2, as described in Reiter et al.,Nucl. Acids Res. (2005) 33 (suppl 1): D671-D674. The CDRs and FRs of theVH regions and VL regions of the antibody clones described herein weredefined according to the international IMGT (ImMunoGeneTics) informationsystem (LeFranc et al., Nucleic Acids Res. (2015) 43 (Databaseissue):D413-22), which uses the IMGT V-DOMAIN numbering rules asdescribed in Lefranc et al., Dev. Comp. Immunol. (2003) 27:55-77.

In some embodiments, the antigen-binding molecule comprises the CDRs ofan antigen-binding molecule which is capable of binding to HER3. In someembodiments, the antigen-binding molecule comprises the FRs of anantigen-binding molecule which is capable of binding to HER3. In someembodiments, the antigen-binding molecule comprises the CDRs and the FRsof an antigen-binding molecule which is capable of binding to HER3. Thatis, in some embodiments the antigen-binding molecule comprises the VHregion and the VL region of an antigen-binding molecule which is capableof binding to HER3.

In some embodiments the antigen-binding molecule comprises a VH regionand a VL region which is, or which is derived from, the VH/VL region ofa HER3-binding antibody clone described herein (i.e. anti-HER3 antibodyclones 10D1_c75, 10D1_c76, 10D1_c77, 10D1_c76v1, 10D1_c78v2, 10D1_11B,10D1_c85v1, 10D1_c85v2, 10D1_c85o1, 10D1_c85o2, 10D1_c87, 10D1_c89,10D1_c90, 10D1_c91, 10D1_c92, 10D1_c93, 10D1, 10A6, 4-35-B2 or 4-35-B4;e.g. 10D1_c89, 10D1_c90 or 10D1_c91; e.g. 10D1_c89).

In some embodiments the antigen-binding molecule comprises a VH regionaccording to one of (1) to (10) below:

(1) (10D1 derived) a VH region incorporating the following CDRs:

-   -   HC-CDR1 having the amino acid sequence of SEQ ID NO:43    -   HC-CDR2 having the amino acid sequence of SEQ ID NO:46    -   HC-CDR3 having the amino acid sequence of SEQ ID NO:51,    -   or a variant thereof in which one or two or three amino acids in        one or more of HC-CDR1, HC-CDR2, or HC-CDR3 are substituted with        another amino acid.

(2) (10D1, 10D1_c75, 10D1_c76, 10D1_c77, 10D1_c78v1, 10D1_c78v2,10D1_11B, 10D1_c87, 10D1_c92, 10D1_c93) a VH region incorporating thefollowing CDRs:

-   -   HC-CDR1 having the amino acid sequence of SEQ ID NO:41    -   HC-CDR2 having the amino acid sequence of SEQ ID NO:44    -   HC-CDR3 having the amino acid sequence of SEQ ID NO:47,    -   or a variant thereof in which one or two or three amino acids in        one or more of HC-CDR1, HC-CDR2, or HC-CDR3 are substituted with        another amino acid.

(3) (10D1_c85v1, 10D1_c85v2) a VH region incorporating the followingCDRs:

-   -   HC-CDR1 having the amino acid sequence of SEQ ID NO:41    -   HC-CDR2 having the amino acid sequence of SEQ ID NO:45    -   HC-CDR3 having the amino acid sequence of SEQ ID NO:47,    -   or a variant thereof in which one or two or three amino acids in        one or more of HC-CDR1, HC-CDR2, or HC-CDR3 are substituted with        another amino acid.

(4) (10D1_c85o1) a VH region incorporating the following CDRs:

-   -   HC-CDR1 having the amino acid sequence of SEQ ID NO:41    -   HC-CDR2 having the amino acid sequence of SEQ ID NO:45    -   HC-CDR3 having the amino acid sequence of SEQ ID NO:49,    -   or a variant thereof in which one or two or three amino acids in        one or more of HC-CDR1, HC-CDR2, or HC-CDR3 are substituted with        another amino acid.

(5) (10D1_c85o2) a VH region incorporating the following CDRS:

-   -   HC-CDR1 having the amino acid sequence of SEQ ID NO:41    -   HC-CDR2 having the amino acid sequence of SEQ ID NO:45    -   HC-CDR3 having the amino acid sequence of SEQ ID NO:50,    -   or a variant thereof in which one or two or three amino acids in        one or more of HC-CDR1, HC-CDR2, or HC-CDR3 are substituted with        another amino acid.

(6) (10D1_c89, 10D1_c90) a VH region incorporating the following CDRs:

-   -   HC-CDR1 having the amino acid sequence of SEQ ID NO:41    -   HC-CDR2 having the amino acid sequence of SEQ ID NO:45    -   HC-CDR3 having the amino acid sequence of SEQ ID NO:48,    -   or a variant thereof in which one or two or three amino acids in        one or more of HC-CDR1, HC-CDR2, or HC-CDR3 are substituted with        another amino acid.

(7) (10D1_c91) a VH region incorporating the following CDRs:

-   -   HC-CDR1 having the amino acid sequence of SEQ ID NO:42    -   HC-CDR2 having the amino acid sequence of SEQ ID NO:45    -   HC-CDR3 having the amino acid sequence of SEQ ID NO:48,    -   or a variant thereof in which one or two or three amino acids in        one or more of HC-CDR1, HC-CDR2, or HC-CDR3 are substituted with        another amino acid.

(8) (10A6) a VH region incorporating the following CDRs:

-   -   HC-CDR1 having the amino acid sequence of SEQ ID NO:158    -   HC-CDR2 having the amino acid sequence of SEQ ID NO:159    -   HC-CDR3 having the amino acid sequence of SEQ ID NO:160,    -   or a variant thereof in which one or two or three amino acids in        one or more of HC-CDR1, HC-CDR2, or HC-CDR3 are substituted with        another amino acid.

(9) (4-35-B2) a VH region incorporating the following CDRs:

-   -   HC-CDR1 having the amino acid sequence of SEQ ID NO:128    -   HC-CDR2 having the amino acid sequence of SEQ ID NO:129    -   HC-CDR3 having the amino acid sequence of SEQ ID NO:130,    -   or a variant thereof in which one or two or three amino acids in        one or more of HC-CDR1, HC-CDR2, or HC-CDR3 are substituted with        another amino acid.

(10) (4-35-B4) a VH region incorporating the following CDRs:

-   -   HC-CDR1 having the amino acid sequence of SEQ ID NO:144    -   HC-CDR2 having the amino acid sequence of SEQ ID NO:145    -   HC-CDR3 having the amino acid sequence of SEQ ID NO:146,    -   or a variant thereof in which one or two or three amino acids in        one or more of HC-CDR1, HC-CDR2, or HC-CDR3 are substituted with        another amino acid.

In some embodiments the antigen-binding molecule comprises a VH regionaccording to one of (11) to (24) below:

(11) (10D1) a VH region incorporating the following FRs:

-   -   HC-FR1 having the amino acid sequence of SEQ ID NO:55    -   HC-FR2 having the amino acid sequence of SEQ ID NO:58    -   HC-FR3 having the amino acid sequence of SEQ ID NO:69    -   HC-FR4 having the amino acid sequence of SEQ ID NO:73,    -   or a variant thereof in which one or two or three amino acids in        one or more of HC-FR1, HC-FR2, HC-FR3, or HC-FR4 are substituted        with another amino acid.

(12) (10D1_c75, 10D1_c92) a VH region incorporating the following FRs:

-   -   HC-FR1 having the amino acid sequence of SEQ ID NO:52    -   HC-FR2 having the amino acid sequence of SEQ ID NO:56    -   HC-FR3 having the amino acid sequence of SEQ ID NO:61    -   HC-FR4 having the amino acid sequence of SEQ ID NO:70,    -   or a variant thereof in which one or two or three amino acids in        one or more of HC-FR1, HC-FR2, HC-FR3, or HC-FR4 are substituted        with another amino add.

(13) (10D1_c76, 10D1_c77, 10D1_c78v1) a VH region incorporating thefollowing FRs:

-   -   HC-FR1 having the amino acid sequence of SEQ ID NO:52    -   HC-FR2 having the amino acid sequence of SEQ ID NO:56    -   HC-FR3 having the amino acid sequence of SEQ ID NO:62    -   HC-FR4 having the amino acid sequence of SEQ ID NO:70,    -   or a variant thereof in which one or two or three amino acids in        one or more of HC-FR1, HC-FR2, HC-FR3, or HC-FR4 are substituted        with another amino acid.

(14) (10D1_c78v2) a VH region incorporating the following FRs:

-   -   HC-FR1 having the amino acid sequence of SEQ ID NO:52    -   HC-FR2 having the amino acid sequence of SEQ ID NO:57    -   HC-FR3 having the amino acid sequence of SEQ ID NO:62    -   HC-FR4 having the amino acid sequence of SEQ ID NO:70,    -   or a variant thereof in which one or two or three amino acids in        one or more of HC-FR1, HC-FR2, HC-FR3, or HC-FR4 are substituted        with another amino acid.

(15) (10D1_11B) a VH region incorporating the following FRs:

-   -   HC-FR1 having the amino acid sequence of SEQ ID NO:224    -   HC-FR2 having the amino acid sequence of SEQ ID NO:60    -   HC-FR3 having the amino acid sequence of SEQ ID NO:83    -   HC-FR4 having the amino acid sequence of SEQ ID NO:70,    -   or a variant thereof in which one or two or three amino acids in        one or more of HC-FR1, HC-FR2, HC-FR3, or HC-FR4 are substituted        with another amino acid.

(16) (10D1_c85v1) a VH region incorporating the following FRs:

-   -   HC-FR1 having the amino acid sequence of SEQ ID NO:52    -   HC-FR2 having the amino acid sequence of SEQ ID NO:56    -   HC-FR3 having the amino acid sequence of SEQ ID NO:64    -   HC-FR4 having the amino acid sequence of SEQ ID NO:70,    -   or a variant thereof in which one or two or three amino acids in        one or more of HC-FR1, HC-FR2, HC-FR3, or HC-FR4 are substituted        with another amino acid.

(17) (10D1_c85v2, 10D1_c85o1, 10D1_c85o2) a VH region incorporating thefollowing FRs:

-   -   HC-FR1 having the amino acid sequence of SEQ ID NO:52    -   HC-FR2 having the amino acid sequence of SEQ ID NO:57    -   HC-FR3 having the amino acid sequence of SEQ ID NO:64    -   HC-FR4 having the amino acid sequence of SEQ ID NO:70,    -   or a variant thereof in which one or two or three amino acids in        one or more of HC-FR1, HC-FR2, HC-FR3, or HC-FR4 are substituted        with another amino acid.

(18) (10D1_c87, 10D1_c93) a VH region incorporating the following FRs:

-   -   HC-FR1 having the amino acid sequence of SEQ ID NO:52    -   HC-FR2 having the amino acid sequence of SEQ ID NO:56    -   HC-FR3 having the amino acid sequence of SEQ ID NO:65    -   HC-FR4 having the amino acid sequence of SEQ ID NO:70,    -   or a variant thereof in which one or two or three amino acids in        one or more of HC-FR1, HC-FR2, HC-FR3, or HC-FR4 are substituted        with another amino acid.

(19) (10D1_c89) a VH region incorporating the following FRs:

-   -   HC-FR1 having the amino acid sequence of SEQ ID NO:53    -   HC-FR2 having the amino acid sequence of SEQ ID NO:59    -   HC-FR3 having the amino acid sequence of SEQ ID NO:66    -   HC-FR4 having the amino acid sequence of SEQ ID NO:71,    -   or a variant thereof in which one or two or three amino acids in        one or more of HC-FR1, HC-FR2, HC-FR3, or HC-FR4 are substituted        with another amino acid.

(20) (10D1_c90) a VH region incorporating the following FRs:

-   -   HC-FR1 having the amino acid sequence of SEQ ID NO:54    -   HC-FR2 having the amino acid sequence of SEQ ID NO:59    -   HC-FR3 having the amino acid sequence of SEQ ID NO:67    -   HC-FR4 having the amino acid sequence of SEQ ID NO:71,    -   or a variant thereof in which one or two or three amino acids in        one or more of HC-FR1, HC-FR2, HC-FR3, or HC-FR4 are substituted        with another amino acid.

(21) (10D1_c91) a VH region incorporating the following FRs:

-   -   HC-FR1 having the amino acid sequence of SEQ ID NO:53    -   HC-FR2 having the amino acid sequence of SEQ ID NO:59    -   HC-FR3 having the amino acid sequence of SEQ ID NO:68    -   HC-FR4 having the amino acid sequence of SEQ ID NO:72,    -   or a variant thereof in which one or two or three amino acids in        one or more of HC-FR1, HC-FR2, HC-FR3, or HC-FR4 are substituted        with another amino acid.

(22) (10A6) a VH region incorporating the following FRs:

-   -   HC-FR1 having the amino acid sequence of SEQ ID NO:161    -   HC-FR2 having the amino acid sequence of SEQ ID NO:162    -   HC-FR3 having the amino acid sequence of SEQ ID NO:163    -   HC-FR4 having the amino acid sequence of SEQ ID NO:73,    -   or a variant thereof in which one or two or three amino acids in        one or more of HC-FR1, HC-FR2, HC-FR3, or HC-FR4 are substituted        with another amino acid.

(23) (4-35-B2) a VH region incorporating the following FRs:

-   -   HC-FR1 having the amino acid sequence of SEQ ID NO:131    -   HC-FR2 having the amino acid sequence of SEQ ID NO:132    -   HC-FR3 having the amino acid sequence of SEQ ID NO:133    -   HC-FR4 having the amino acid sequence of SEQ ID NO:134,    -   or a variant thereof in which one or two or three amino acids in        one or more of HC-FR1, HC-FR2, HC-FR3, or HC-FR4 are substituted        with another amino acid.

(24) (4-35-B4) a VH region incorporating the following FRs:

-   -   HC-FR1 having the amino acid sequence of SEQ ID NO:147    -   HC-FR2 having the amino acid sequence of SEQ ID NO:148    -   HC-FR3 having the amino acid sequence of SEQ ID NO:149    -   HC-FR4 having the amino acid sequence of SEQ ID NO:73,    -   or a variant thereof in which one or two or three amino acids in        one or more of HC-FR1, HC-FR2, HC-FR3, or HC-FR4 are substituted        with another amino acid.

In some embodiments the antigen-binding molecule comprises a VH regioncomprising the CDRs according to one of (1) to (10) above, and the FRsaccording to one of (11) to (24) above.

In some embodiments the antigen-binding molecule comprises a VH regionaccording to one of (25) to (41) below:

(25) a VH region comprising the CDRs according to (1) and the FRsaccording to (11), (12), (13), (14), (15), (16), (17), (18), (19), (20)or (21).

(26) a VH region comprising the CDRs according to (2) and the FRsaccording to (11).

(27) a VH region comprising the CDRs according to (2) and the FRsaccording to (12).

(28) a VH region comprising the CDRs according to (2) and the FRsaccording to (13).

(29) a VH region comprising the CDRs according to (2) and the FRsaccording to (14).

(30) a VH region comprising the CDRs according to (2) and the FRsaccording to (15).

(31) a VH region comprising the CDRs according to (2) and the FRsaccording to (18).

(32) a VH region comprising the CDRs according to (3) and the FRsaccording to (16).

(33) a VH region comprising the CDRs according to (3) and the FRsaccording to (17).

(34) a VH region comprising the CDRs according to (4) and the FRsaccording to (17).

(35) a VH region comprising the CDRs according to (5) and the FRsaccording to (17).

(36) a VH region comprising the CDRs according to (6) and the FRsaccording to (19).

(37) a VH region comprising the CDRs according to (6) and the FRsaccording to (20).

(38) a VH region comprising the CDRs according to (7) and the FRsaccording to (21).

(39) a VH region comprising the CDRs according to (8) and the FRsaccording to (22).

(40) a VH region comprising the CDRs according to (9) and the FRsaccording to (23).

(41) a VH region comprising the CDRs according to (10) and the FRsaccording to (24).

In some embodiments the antigen-binding molecule comprises a VH regionaccording to one of (42) to (61) below:

(42) a VH region comprising an amino acid sequence having at least 70%sequence identity more preferably one of at least 75%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%, sequence identity to the amino acid sequence of SEQ ID NO:24.

(43) a VH region comprising an amino acid sequence having at least 70%sequence identity more preferably one of at least 75%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%, sequence identity to the amino acid sequence of SEQ ID NO:25.

(44) a VH region comprising an amino acid sequence having at least 70%sequence identity more preferably one of at least 75%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%, sequence identity to the amino acid sequence of SEQ ID NO:26.

(45) a VH region comprising an amino acid sequence having at least 70%sequence identity more preferably one of at least 75%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%, sequence identity to the amino acid sequence of SEQ ID NO:27.

(46) a VH region comprising an amino acid sequence having at least 70%sequence identity more preferably one of at least 75%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%, sequence identity to the amino acid sequence of SEQ ID NO:28.

(47) a VH region comprising an amino acid sequence having at least 70%sequence identity more preferably one of at least 75%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%, sequence identity to the amino acid sequence of SEQ ID NO:29.

(48) a VH region comprising an amino acid sequence having at least 70%sequence identity more preferably one of at least 75%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%, sequence identity to the amino acid sequence of SEQ ID NO:30.

(49) a VH region comprising an amino acid sequence having at least 70%sequence identity more preferably one of at least 75%, 80%, 85%, 86%,87%, 8830, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%, sequence identity to the amino acid sequence of SEQ ID NO:31.

(50) a VH region comprising an amino acid sequence having at least 70%sequence identity more preferably one of at least 75%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%, sequence identity to the amino acid sequence of SEQ ID NO:32.

(51) a VH region comprising an amino acid sequence having at least 70%sequence identity more preferably one of at least 75%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%, sequence identity to the amino acid sequence of SEQ ID NO:33.

(52) a VH region comprising an amino acid sequence having at least 70%sequence identity more preferably one of at least 75%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%, sequence identity to the amino acid sequence of SEQ ID NO:34.

(53) a VH region comprising an amino acid sequence having at least 70%sequence identity more preferably one of at least 75%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%, sequence identity to the amino acid sequence of SEQ ID NO:35.

(54) a VH region comprising an amino acid sequence having at least 70%sequence identity more preferably one of at least 75%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%, sequence identity to the amino acid sequence of SEQ ID NO:36.

(55) a VH region comprising an amino acid sequence having at least 70%sequence identity more preferably one of at least 75%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%, sequence identity to the amino acid sequence of SEQ ID NO:37.

(56) a VH region comprising an amino acid sequence having at least 70%sequence identity more preferably one of at least 75%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%, sequence identity to the amino acid sequence of SEQ ID NO:38.

(57) a VH region comprising an amino acid sequence having at least 70%sequence identity more preferably one of at least 75%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%, sequence identity to the amino acid sequence of SEQ ID NO:39.

(58) a VH region comprising an amino acid sequence having at least 70%sequence identity more preferably one of at least 75%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%, sequence identity to the amino acid sequence of SEQ ID NO:40.

(59) a VH region comprising an amino acid sequence having at least 70%sequence identity more preferably one of at least 75%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%, sequence identity to the amino acid sequence of SEQ ID NO:127.

(60) a VH region comprising an amino acid sequence having at least 70%sequence identity more preferably one of at least 75%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%, sequence identity to the amino acid sequence of SEQ ID NO:143.

(61) a VH region comprising an amino acid sequence having at least 70%sequence identity more preferably one of at least 75%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%, sequence identity to the amino acid sequence of SEQ ID NO:157.

In some embodiments the antigen-binding molecule comprises a VL regionaccording to one of (62) to (71) below:

(62) (10D1 derived) a VL region incorporating the following CDRs:

-   -   LC-CDR1 having the amino acid sequence of SEQ ID NO:91    -   LC-CDR2 having the amino acid sequence of SEQ ID NO:94    -   LC-CDR3 having the amino acid sequence of SEQ ID NO:99;    -   or a variant thereof in which one or two or three amino acids in        one or more of LC-CDR1, LC-CDR2 or LC-CDR3 are substituted with        another amino acid.

(63) (10D1, 10D1_c75, 10D1_c78v1, 10D1_c78v2, 10D1_11B, 10D1_c87,10D1_c89, 10D1_c91, 10D1_c93) a VL region incorporating the followingCDRs:

-   -   LC-CDR1 having the amino acid sequence of SEQ ID NO:88    -   LC-CDR2 having the amino acid sequence of SEQ ID NO:92    -   LC-CDR3 having the amino acid sequence of SEQ ID NO:95;    -   or a variant thereof in which one or two or three amino acids in        one or more of LC-CDR1, LC-CDR2 or LC-CDR3 are substituted with        another amino acid.

(64) (10D1_c76) a VL region incorporating the following CDRs:

-   -   LC-CDR1 having the amino acid sequence of SEQ ID NO:89    -   LC-CDR2 having the amino acid sequence of SEQ ID NO:92    -   LC-CDR3 having the amino acid sequence of SEQ ID NO:95;    -   or a variant thereof in which one or two or three amino acids in        one or more of LC-CDR1, LC-CDR2 or LC-CDR3 are substituted with        another amino acid.

(65) (10D1_c77) a VL region incorporating the following CDRs:

-   -   LC-CDR1 having the amino acid sequence of SEQ ID NO:90    -   LC-CDR2 having the amino acid sequence of SEQ ID NO:92    -   LC-CDR3 having the amino acid sequence of SEQ ID NO:96;    -   or a variant thereof in which one or two or three amino acids in        one or more of LC-CDR1, LC-CDR2 or LC-CDR3 are substituted with        another amino acid.

(66) (10D1_c85v1, 10D1_c85v2, 10D1_c85o1, 10D1_c85o2) a VL regionincorporating the following CDRs:

-   -   LC-CDR1 having the amino acid sequence of SEQ ID NO:88    -   LC-CDR2 having the amino acid sequence of SEQ ID NO:93    -   LC-CDR3 having the amino acid sequence of SEQ ID NO:95;    -   or a variant thereof in which one or two or three amino acids in        one or more of LC-CDR1 LC-CDR2 or LC-CDR3 are substituted with        another amino acid.

(67) (10D1_c90) a VL region incorporating the following CDRs:

-   -   LC-CDR1 having the amino acid sequence of SEQ ID NO:88    -   LC-CDR2 having the amino acid sequence of SEQ ID NO:92    -   LC-CDR3 having the amino acid sequence of SEQ ID NO:97;    -   or a variant thereof in which one or two or three amino acids in        one or more of LC-CDR1, LC-CDR2 or LC-CDR3 are substituted with        another amino acid.

(68) (10D1_c92) a VL region incorporating the following CDRs:

-   -   LC-CDR1 having the amino acid sequence of SEQ ID NO:88    -   LC-CDR2 having the amino acid sequence of SEQ ID NO:92    -   LC-CDR3 having the amino acid sequence of SEQ ID NO:98;    -   or a variant thereof in which one or two or three amino acids in        one or more of LC-CDR1, LC-CDR2 or LC-CDR3 are substituted with        another amino acid.

(69) (10A6) a VL region incorporating the following CDRs;

-   -   LC-CDR1 having the amino acid sequence of SEQ ID NO:165    -   LC-CDR2 having the amino acid sequence of SEQ ID NO:166    -   LC-CDR3 having the amino acid sequence of SEQ ID NO:167;    -   or a variant thereof in which one or two or three amino acids in        one or more of LC-CDR1 LC-CDR2 or LC-CDR3 are substituted with        another amino acid.

(70) (4-35-B2) a VL region incorporating the following CDRs:

-   -   LC-CDR1 having the amino acid sequence of SEQ ID NO:136    -   LC-CDR2 having the amino acid sequence of SEQ ID NO:137    -   LC-CDR3 having the amino acid sequence of SEQ ID NO:138,    -   or a variant thereof in which one or two or three amino acids in        one or more of LC-CDR1, LC-CDR2 or LC-CDR3 are substituted with        another amino acid.

(71) (4-35-B4) a VL region incorporating the following CDRs:

-   -   LC-CDR1 having the amino acid sequence of SEQ ID NO:151    -   LC-CDR2 having the amino acid sequence of SEQ ID NO:152    -   LC-CDR3 having the amino acid sequence of SEQ ID NO:153;    -   or a variant thereof in which one or two or three amino acids in        one or more of LC-CDR1. LC-CDR2 or LC-CDR3 are substituted with        another amino acid.

In some embodiments the antigen-binding molecule comprises a VL regionaccording to one of (72) to (86) below;

(72) (10D1) a VL region incorporating the following FRs:

-   -   LC-FR1 having the amino acid sequence of SEQ ID NO:106    -   LC-FR2 having the amino acid sequence of SEQ ID NO:113    -   LC-FR3 having the amino acid sequence of SEQ ID NO:123    -   LC-FR4 having the amino acid sequence of SEQ ID NO:126,    -   or a variant thereof in which one or two or three amino acids in        one or more of LC-FR1, LC-FR2, LC-FR3, or LC-FR4 are substituted        with another amino acid.

(73) (10D1_c75) a VL region incorporating the following FRs;

-   -   LC-FR1 having the amino acid sequence of SEQ ID NO:100    -   LC-FR2 having the amino acid sequence of SEQ ID NO:107    -   LC-FR3 having the amino acid sequence of SEQ ID NO:114    -   LC-FR4 having the amino acid sequence of SEQ ID NO:124,    -   or a variant thereof in which one or two or three amino acids in        one or more of LC-FR1, LC-FR2, LC-FR3, or LC-FR4 are substituted        with another amino acid.

(74) (10D1_c76) a VL region incorporating the following FRs:

-   -   LC-FR1 having the amino acid sequence of SEQ ID NO:101    -   LC-FR2 having the amino acid sequence of SEQ ID NO:108    -   LC-FR3 having the amino acid sequence of SEQ ID NO:115    -   LC-FR4 having the amino acid sequence of SEQ ID NO:124,    -   or a variant thereof in which one or two or three amino acids in        one or more of LC-FR1, LC-FR2, LC-FR3, or LC-FR4 are substituted        with another amino acid.

(75) (10D1_c77) a VL region incorporating the following FRs:

-   -   LC-FR1 having the amino acid sequence of SEQ ID NO:102    -   LC-FR2 having the amino acid sequence of SEQ ID NO:108    -   LC-FR3 having the amino acid sequence of SEQ ID NO:116    -   LC-FR4 having the amino acid sequence of SEQ ID NO:124,    -   or a variant thereof in which one or two or three amino acids in        one or more of LC-FR1, LC-FR2, LC-FR3, or LC-FR4 are substituted        with another amino acid.

(76) (10D1_c78v1, 10D1_c78v2, 10D1_11B) a VL region incorporating thefollowing FRs:

-   -   LC-FR1 having the amino acid sequence of SEQ ID NO:103    -   LC-FR2 having the amino acid sequence of SEQ ID NO:108    -   LC-FR3 having the amino acid sequence of SEQ ID NO:117    -   LC-FR4 having the amino acid sequence of SEQ ID NO:124,    -   or a variant thereof in which one or two or three amino acids in        one or more of LC-FR1, LC-FR2, LC-FR3, or LC-FR4 are substituted        with another amino acid.

(77) (10D1_c85v1, 10D1_c85v2, 10D1_c85o1, 10D1_c85o2) a VL regionincorporating the following FRs:

-   -   LC-FR1 having the amino acid sequence of SEQ ID NO:103    -   LC-FR2 having the amino acid sequence of SEQ ID NO:108    -   LC-FR3 having the amino acid sequence of SEQ ID NO:118    -   LC-FR4 having the amino acid sequence of SEQ ID NO:124,    -   or a variant thereof in which one or two or three amino acids in        one or more of LC-FR1, LC-FR2, LC-FR3, or LC-FR4 are substituted        with another amino acid.

(78) (10D1_c87) a VL region incorporating the following FRs:

-   -   LC-FR1 having the amino acid sequence of SEQ ID NO:103    -   LC-FR2 having the amino acid sequence of SEQ ID NO:109    -   LC-FR3 having the amino acid sequence of SEQ ID NO:119    -   LC-FR4 having the amino acid sequence of SEQ ID NO:124,    -   or a variant thereof in which one or two or three amino acids in        one or more of LC-FR1, LC-FR2, LC-FR3, or LC-FR4 are substituted        with another amino acid.

(79) (10D1_c89) a VL region incorporating the following FRs:

-   -   LC-FR1 having the amino acid sequence of SEQ ID NO:104    -   LC-FR2 having the amino acid sequence of SEQ ID NO:110    -   LC-FR3 having the amino acid sequence of SEQ ID NO:120    -   LC-FR4 having the amino acid sequence of SEQ ID NO:125,    -   or a variant thereof in which one or two or three amino acids in        one or more of LC-FR1, LC-FR2, LC-FR3, or LC-FR4 are substituted        with another amino acid.

(80) (10D1_c90) a VL region incorporating the following FRs:

-   -   LC-FR1 having the amino acid sequence of SEQ ID NO:105    -   LC-FR2 having the amino acid sequence of SEQ ID NO:110    -   LC-FR3 having the amino acid sequence of SEQ ID NO:121    -   LC-FR4 having the amino acid sequence of SEQ ID NO:124,    -   or a variant thereof in which one or two or three amino acids in        one or more of LC-FR1, LC-FR2, LC-FR3, or LC-FR4 are substituted        with another amino acid.

(81) (10D1_c91) a VL region incorporating the following FRs:

-   -   LC-FR1 having the amino acid sequence of SEQ ID NO:104    -   LC-FR2 having the amino acid sequence of SEQ ID NO:111    -   LC-FR3 having the amino acid sequence of SEQ ID NO:122    -   LC-FR4 having the amino acid sequence of SEQ ID NO:125,    -   or a variant thereof in which one or two or three amino acids in        one or more of LC-FR1 LC-FR2, LC-FR3, or LC-FR4 are substituted        with another amino acid.

(82) (10D1_c92) a VL region incorporating the following FRs:

-   -   LC-FR1 having the amino acid sequence of SEQ ID NO:100    -   LC-FR2 having the amino acid sequence of SEQ ID NO:112    -   LC-FR3 having the amino acid sequence of SEQ ID NO:114    -   LC-FR4 having the amino acid sequence of SEQ ID NO:124,    -   or a variant thereof in which one or two or three amino acids in        one or more of LC-FR1, LC-FR2, LC-FR3, or LC-FR4 are substituted        with another amino acid.

(83) (10D1_c93) a VL region incorporating the following FRs:

-   -   LC-FR1 having the amino acid sequence of SEQ ID NO:103    -   LC-FR2 having the amino acid sequence of SEQ ID NO:108    -   LC-FR3 having the amino acid sequence of SEQ ID NO:119    -   LC-FR4 having the amino acid sequence of SEQ ID NO:124,    -   or a variant thereof in which one or two or three amino acids in        one or more of LC-FR1, LC-FR2, LC-FR3, or LC-FR4 are substituted        with another amino acid.

(84) (10A6) a VL region incorporating the following FRs:

-   -   LC-FR1 having the amino acid sequence of SEQ ID NO:168    -   LC-FR2 having the amino acid sequence of SEQ ID NO:169    -   LC-FR3 having the amino acid sequence of SEQ ID NO:170    -   LC-FR4 having the amino acid sequence of SEQ ID NO:142,    -   or a variant thereof in which one or two or three amino acids in        one or more of LC-FR1, LC-FR2, LC-FR3, or LC-FR4 are substituted        with another amino acid.

(85) (4-35-B2) a VL region incorporating the following FRs:

-   -   LC-FR1 having the amino acid sequence of SEQ ID NO:139    -   LC-FR2 having the amino acid sequence of SEQ ID NO:140    -   LC-FR3 having the amino acid sequence of SEQ ID NO:141    -   LC-FR4 having the amino acid sequence of SEQ ID NO:142,    -   or a variant thereof in which one or two or three amino acids in        one or more of LC-FR1, LC-FR2, LC-FR3, or LC-FR4 are substituted        with another amino acid.

(86) (4-35-B4) a VL region incorporating the following FRs:

-   -   LC-FR1 having the amino acid sequence of SEQ ID NO:154    -   LC-FR2 having the amino acid sequence of SEQ ID NO:155    -   LC-FR3 having the amino acid sequence of SEQ ID NO:156    -   LC-FR4 having the amino acid sequence of SEQ ID NO:142,    -   or a variant thereof in which one or two or three amino acids in        one or more of LC-FR1. LC-FR2, LC-FR3, or LC-FR4 are substituted        with another amino acid.

In some embodiments the antigen-binding molecule comprises a VL regioncomprising the CDRs according to one of (62) to (71) above, and the FRsaccording to one of (72) to (86) above.

In some embodiments the antigen-binding molecule comprises a VL regionaccording to one of (87) to (102) below:

(87) a VL region comprising the CDRs according to (62) and the FRsaccording to (72), (73), (74), (75), (76), (77), (78), (79), (80), (81),(82), or (83).

(88) a VL region comprising the CDRs according to (63) and the FRsaccording to (72).

(89) a VL region comprising the CDRs according to (63) and the FRsaccording to (73).

(90) a VL region comprising the CDRs according to (63) and the FRsaccording to (76).

(91) a VL region comprising the CDRs according to (63) and the FRsaccording to (78).

(92) a VL region comprising the CDRs according to (63) and the FRsaccording to (79).

(93) a VL region comprising the CDRs according to (63) and the FRsaccording to (81).

(94) a VL region comprising the CDRs according to (63) and the FRsaccording to (83).

(95) a VL region comprising the CDRs according to (64) and the FRsaccording to (74).

(96) a VL region comprising the CDRs according to (65) and the FRsaccording to (75).

(97) a VL region comprising the CDRs according to (66) and the FRsaccording to (77).

(98) a VL region comprising the CDRs according to (67) and the FRsaccording to (80).

(99) a VL region comprising the CDRs according to (68) and the FRsaccording to (82).

(100) a VL region comprising the CDRs according to (69) and the FRsaccording to (84).

(101) a VL region comprising the CDRs according to (70) and the FRsaccording to (85).

(102) a VL region comprising the CDRs according to (71) and the FRsaccording to (86).

In some embodiments the antigen-binding molecule comprises a VL regionaccording to one of (103) to (119) below:

(103) a VL region comprising an amino acid sequence having at least 70%sequence identity more preferably one of at least 75%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%, sequence identity to the amino acid sequence of SEQ ID NO:74.

(104) a VL region comprising an amino acid sequence having at least 70%sequence identity more preferably one of at least 75%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%, sequence identity to the amino acid sequence of SEQ ID NO:75.

(105) a VL region comprising an amino acid sequence having at least 70%sequence identity more preferably one of at least 75%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%, sequence identity to the amino acid sequence of SEQ ID NO:76.

(106) a VL region comprising an amino acid sequence having at least 70%sequence identity more preferably one of at least 75%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%, sequence identity to the amino acid sequence of SEQ ID NO:77.

(107) a VL region comprising an amino acid sequence having at least 70%sequence identity more preferably one of at least 75%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 93%, 99%, or100%, sequence identity to the amino acid sequence of SEQ ID NO:78.

(108) a VL region comprising an amino acid sequence having at least 70%sequence identity more preferably one of at least 75%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%, sequence identity to the amino acid sequence of SEQ ID NO:79.

(109) a VL region comprising an amino acid sequence having at least 70%sequence identity more preferably one of at least 75%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%, sequence identity to the amino acid sequence of SEQ ID NO:80.

(110) a VL region comprising an amino acid sequence having at least 70%sequence identity more preferably one of at least 75%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%, sequence identity to the amino acid sequence of SEQ ID NO:81.

(111) a VL region comprising an amino acid sequence having at least 70%sequence identity more preferably one of at least 75%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%, sequence identity to the amino acid sequence of SEQ ID NO:82.

(112) a VL region comprising an amino acid sequence having at least 70%sequence identity more preferably one of at least 75%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%, sequence identity to the amino acid sequence of SEQ ID NO:83.

(113) a VL region comprising an amino acid sequence having at least 70%sequence identity more preferably one of at least 75%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%, sequence identity to the amino acid sequence of SEQ ID NO:84.

(114) a VL region comprising an amino acid sequence having at least 70%sequence identity more preferably one of at least 75%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%, sequence identity to the amino acid sequence of SEQ ID NO:85.

(115) a VL region comprising an amino acid sequence having at least 70%sequence identity more preferably one of at least 75%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%, sequence identity to the amino acid sequence of SEQ ID NO:86.

(116) a VL region comprising an amino acid sequence having at least 70%sequence identity more preferably one of at least 75%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%, sequence identity to the amino acid sequence of SEQ ID NO:87.

(117) a VL region comprising an amino acid sequence having at least 70%sequence identity more preferably one of at least 75%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%, sequence identity to the amino acid sequence of SEQ ID NO:135.

(118) a VL region comprising an amino acid sequence having at least 70%sequence identity more preferably one of at least 75%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%, sequence identity to the amino acid sequence of SEQ ID NO:150.

(119) a VL region comprising an amino acid sequence having at least 70%sequence identity more preferably one of at least 75%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%, sequence identity to the amino acid sequence of SEQ ID NO:164.

In some embodiments the antigen-binding molecule comprises a VH regionaccording to any one of (1) to (61) above, and a VL region according toany one of (62) to (119) above.

In embodiments in accordance with the present invention in which one ormore amino acids are substituted with another amino acid, thesubstitutions may be conservative substitutions, for example accordingto the following Table. In some embodiments, amino acids in the sameblock in the middle column are substituted. In some embodiments, aminoacids in the same line in the rightmost column are substituted:

ALIPHATIC Non-polar G A P I L V Polar - uncharged C S T M N Q Polar -charged D E K R AROMATIC H F W Y

In some embodiments, substitution(s) may be functionally conservative.That is, in some embodiments the substitution may not affect (or may notsubstantially affect) one or more functional properties (e.g. targetbinding) of the antigen-binding molecule comprising the substitution ascompared to the equivalent unsubstituted molecule.

The VH and VL region of an antigen-binding region of an antibodytogether constitute the Fv region. In some embodiments, theantigen-binding molecule according to the present invention comprises,or consists of, an Fv region which binds to HER3. In some embodimentsthe VH and VL regions of the Fv are provided as single polypeptidejoined by a linker region, i.e. a single chain Fv (scFv).

In some embodiments the antigen-binding molecule of the presentinvention comprises one or more regions of an immunoglobulin heavy chainconstant sequence. In some embodiments the immunoglobulin heavy chainconstant sequence is, or is derived from, the heavy chain constantsequence of an IgG (e.g. IgG1, IgG2, IgG3, IgG4), IgA (e.g. IgA1, IgA2),IgD, IgE or IgM.

In some embodiments the immunoglobulin heavy chain constant sequence ishuman immunoglobulin G 1 constant (IGHG1; UniProt, P01857-1, v1; SEQ IDNO:171). Positions 1 to 98 of SEQ ID NO:171 form the CH1 region (SEQ IDNO:172). Positions 99 to 110 of SEQ ID NO:171 form a hinge regionbetween CH1 and CH2 regions (SEQ ID NO:173). Positions 111 to 223 of SEQID NO:171 form the CH2 region (SEQ ID NO:174). Positions 224 to 330 ofSEQ ID NO:171 form the CH3 region (SEQ ID NO:175).

The exemplified antigen-binding molecules may be prepared usingpFUSE-CHIg-hG1, which comprises the substitutions D356E, L358M(positions numbered according to EU numbering) in the CH3 region. Theamino acid sequence of the CH3 region encoded by pFUSE-CHIg-hG1 is shownin SEQ ID NO:176. It will be appreciated that CH3 regions may beprovided with further substitutions in accordance with modification toan Fc region of the antigen-binding molecule as described herein.

In some embodiments a CH1 region comprises or consists of the sequenceof SEQ ID NO:172, or a sequence having at least 60%, preferably one of70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or100% amino acid sequence identity to the amino acid sequence of SEQ IDNO:172. In some embodiments a CH1-CH2 hinge region comprises or consistsof the sequence of SEQ ID NO:173, or a sequence having at least 60%,preferably one of 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or 100% amino acid sequence identity to the amino acidsequence of SEQ ID NO:173. In some embodiments a CH2 region comprises orconsists of the sequence of SEQ ID NO:174, or a sequence having at least60%, preferably one of 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% amino acid sequence identity to the aminoacid sequence of SEQ ID NO:174. In some embodiments a CH3 regioncomprises or consists of the sequence of SEQ ID NO:175 or 176, or asequence having at least 60%, preferably one of 70%, 75%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequenceidentity to the amino acid sequence of SEQ ID NO:175 or 176.

In some embodiments the antigen-binding molecule of the presentinvention comprises one or more regions of an immunoglobulin light chainconstant sequence. In some embodiments the immunoglobulin light chainconstant sequence is human immunoglobulin kappa constant (IGKC; Cκ;UniProt: P01834-1, v2; SEQ ID NO:177). In some embodiments theimmunoglobulin light chain constant sequence is a human immunoglobulinlambda constant (IGLC: Cλ), e.g. IGLC1, IGLC2, IGLC3, IGLC6 or IGLC7. Insome embodiments a CL region comprises or consists of the sequence ofSEQ ID NO:177, or a sequence having at least 60%, preferably one of 70%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%amino acid sequence identity to the amino acid sequence of SEQ IDNO:177.

The VL and light chain constant (CL) region, and the VH region and heavychain constant 1 (CH1) region of an antigen-binding region of anantibody together constitute the Fab region. In some embodiments theantigen-binding molecule comprises a Fab region comprising a VH, a CH1,a VL and a CL (e.g. Cκ or Cλ). In some embodiments the Fab regioncomprises a polypeptide comprising a VH and a CH1 (e.g. a VH-CH1 fusionpolypeptide), and a polypeptide comprising a VL and a CL (e.g. a VL-CLfusion polypeptide). In some embodiments the Fab region comprises apolypeptide comprising a VH and a CL (e.g. a VH-CL fusion polypeptide)and a polypeptide comprising a VL and a CH (e.g. a VL-CH1 fusionpolypeptide), that is, in some embodiments the Fab region is a CrossFabregion. In some embodiments the VH, CH1, VL and CL regions of the Fab orCrossFab are provided as single polypeptide joined by linker regions,i.e. as a single chain Fab (scFab) or a single chain CrossFab(scCrossFab).

In some embodiments, the antigen-binding molecule of the presentinvention comprises, or consists of, a Fab region which binds to HER3.

In some embodiments, the antigen-binding molecule described hereincomprises, or consists of, a whole antibody which binds to HER3. As usedherein, “whole antibody” refers to an antibody having a structure whichis substantially similar to the structure of an immunoglobulin (Ig).Different kinds of immunoglobulins and their structures are describede.g. in Schroeder and Cavacini J Allergy Clin Immunol. (2010) 125(202):S41-S52, which is hereby incorporated by reference in its entirety.

Immunoglobulins of type G (i.e. IgG) are ˜150 kDa glycoproteinscomprising two heavy chains and two light chains. From N- to C-terminus,the heavy chains comprise a VH followed by a heavy chain constant regioncomprising three constant domains (CH1, CH2, and CH3), and similarly thelight chain comprise a VL followed by a CL. Depending on the heavychain, immunoglobulins may be classed as IgG (e.g. IgG1, IgG2, IgG3,IgG4), IgA (e.g. IgA1, IgA2), IgE, or IgM. The light chain may be kappa(κ) or lambda (λ).

In some embodiments, the antigen-binding molecule described hereincomprises, or consists of, an IgG (e.g. IgG1, IgG2, IgG3, IgG4), IgA(e.g. IgA1, IgA2), IgD, IgE, or IgM which binds to HER3.

In some embodiments, the antigen-binding molecule of the presentinvention is at least monovalent binding for HER3. Binding valencyrefers to the number of binding sites in an antigen-binding molecule fora given antigenic determinant. Accordingly, in some embodiments theantigen-binding molecule comprises at least one binding site for HER3.

In some embodiments the antigen-binding molecule comprises more than onebinding site for HER3, e.g. 2, 3 or 4 binding sites. The binding sitesmay be the same or different. In some embodiments the antigen-bindingmolecule is e.g. bivalent, trivalent or tetravalent for HER3.

Aspects of the present invention relate to multispecific antigen-bindingmolecules. By “multispecific” it is meant that the antigen-bindingmolecule displays specific binding to more than one target. In someembodiments the antigen-binding molecule is a bispecific antigen-bindingmolecule. In some embodiments the antigen-binding molecule comprises atleast two different antigen-binding domains (i.e. at least twoantigen-binding domains, e.g. comprising non-identical VHs and VLs).

In some embodiments the antigen-binding molecule binds to HER3 andanother target (e.g. an antigen other than HER3), and so is at leastbispecific. The term “bispecific” means that the antigen-bindingmolecule is able to bind specifically to at least two distinct antigenicdeterminants.

It will be appreciated that an antigen-binding molecule according to thepresent invention (e.g. a multispecific antigen-binding molecule) maycomprise antigen-binding molecules capable of binding to the targets forwhich the antigen-binding molecule is specific. For example, anantigen-binding molecule which is capable of binding to HER3 and anantigen other than HER3 may comprise: (i) an antigen-binding moleculewhich is capable of binding to HER3, and (ii) an antigen-bindingmolecule which is capable of binding to an antigen other than HER3.

It will also be appreciated that an antigen-binding molecule accordingto the present invention (e.g. a multispecific antigen-binding molecule)may comprise antigen-binding polypeptides or antigen-binding polypeptidecomplexes capable of binding to the targets for which theantigen-binding molecule is specific. For example, an antigen-bindingmolecule according to the invention may comprise e.g. (i) anantigen-binding polypeptide complex capable of binding to HER3,comprising a light chain polypeptide (comprising the structure VL-CL)and a heavy chain polypeptide (comprising the structure VH-CH1-CH2-CH3),and (ii) an antigen-binding polypeptide complex capable of binding to anantigen other than HER3, comprising a light chain polypeptide(comprising the structure VL-CL) and a heavy chain polypeptide(comprising the structure VH-CH1-CH2-CH3).

In some embodiments, a component antigen-binding molecule of a largerantigen-binding molecule (e.g. a multispecific antigen-biding molecule)may be referred to e.g. as an “antigen-binding domain” or“antigen-binding region” of the larger antigen-binding molecule.

In some embodiments the antigen-binding molecule comprises anantigen-binding molecule capable of binding to HER3, and anantigen-binding molecule capable of binding to an antigen other thanHER3. In some embodiments, the antigen other than HER3 is an immune cellsurface molecule. In some embodiments, the antigen other than HER3 is acancer cell antigen. In some embodiments the antigen other than HER3 isa receptor molecule, e.g. a cell surface receptor. In some embodimentsthe antigen other than HER3 is a cell signalling molecule, e.g. acytokine, chemokine, interferon, interleukin or lymphokine. In someembodiments the antigen other than HER3 is a growth factor or a hormone.

A cancer cell antigen is an antigen which is expressed or over-expressedby a cancer cell. A cancer cell antigen may be any peptide polypeptide,glycoprotein, lipoprotein, glycan, glycolipid, lipid, or fragmentthereof. A cancer cell antigen's expression may be associated with acancer. A cancer cell antigen may be abnormally expressed by a cancercell (e.g. the cancer cell antigen may be expressed with abnormallocalisation), or may be expressed with an abnormal structure by acancer cell. A cancer cell antigen may be capable of eliciting an immuneresponse. In some embodiments, the antigen is expressed at the cellsurface of the cancer cell (i.e. the cancer cell antigen is a cancercell surface antigen). In some embodiments, the part of the antigenwhich is bound by the antigen-binding molecule described herein isdisplayed on the external surface of the cancer cell (i.e. isextracellular). The cancer cell antigen may be a cancer-associatedantigen. In some embodiments the cancer cell antigen is an antigen whoseexpression is associated with the development, progression or severityof symptoms of a cancer. The cancer-associated antigen may be associatedwith the cause or pathology of the cancer, or may be expressedabnormally as a consequence of the cancer. In some embodiments, thecancer cell antigen is an antigen whose expression is upregulated (e.g.at the RNA and/or protein level) by cells of a cancer, e.g. as comparedto the level of expression by comparable noncancerous cells (e.g.non-cancerous cells derived from the same tissue/cell type). In someembodiments, the cancer-associated antigen may be preferentiallyexpressed by cancerous cells, and not expressed by comparablenon-cancerous cells (e.g. non-cancerous cells derived from the sametissue/cell type). In some embodiments, the cancer-associated antigenmay be the product of a mutated oncogene or mutated tumor suppressorgene. In some embodiments, the cancer-associated antigen may be theproduct of an overexpressed cellular protein, a cancer antigen producedby an oncogenic virus, an oncofetal antigen, or a cell surfaceglycolipid or glycoprotein.

In some embodiments the antigen other than HER3 is an antigen expressedby cells of a HER3-associated cancer. A HER3-associated cancer may be acancer expressing HER3 (e.g. expressing HER3 protein at the cellsurface): such cancers may be referred to as “HER3-positive” cancers.HER3-associated cancers include cancers for which HER3 gene/proteinexpression is a risk factor for, and/or is positively associated with,the onset, development, progression or severity of symptoms of thecancer, and/or metastasis. HER3-associated cancers include thosedescribed in Zhang et al., Acta Biochimica et Biophysica Sinica (2016)48(1):39-48 and Sithanandam and Anderson Cancer Gene Ther (2003)15(7):413-448, both of which are hereby incorporated by reference intheir entirety. In some embodiments a HER3-associated cancer may be alung cancer (e.g. NSCLC), melanoma, breast cancer, pancreatic cancer,prostate cancer, ovarian cancer, gastric cancer, colon cancer or oralcavity cancer.

An immune cell surface molecule may be any peptide/polypeptide,glycoprotein, lipoprotein, glycan, glycolipid, lipid, or fragmentthereof expressed at or on the cell surface of an immune cell. In someembodiments, the part of the immune cell surface molecule which is boundby the antigen-binding molecule of the present invention is on theexternal surface of the immune cell (i.e. is extracellular). The immunecell surface molecule may be expressed at the cell surface of any immunecell. In some embodiments, the immune cell may be a cell ofhematopoietic origin, e.g. a neutrophil, eosinophil, basophil, dendriticcell, lymphocyte, or monocyte. The lymphocyte may be e.g. a T cell, Bcell, natural killer (NK) cell, NKT cell or innate lymphoid cell (ILO),or a precursor thereof (e.g. a thymocyte or pre-B cell). In someembodiments the immune cell surface molecule may be a costimulatorymolecule (e.g. CD28, OX40, 4-1BB, ICOS or CD27) or a ligand thereof. Insome embodiments the immune cell surface molecule may be a checkpointmolecule (e.g. PD-1, CTLA-4, LAG-3, TIM-3, VISTA, TIGIT or BTLA) or aligand thereof.

Multispecific antigen-binding molecules according to the invention maybe provided in any suitable format, such as those formats described indescribed in Brinkmann and Kontermann MAbs (2017) 9(2): 182-212, whichis hereby incorporated by reference in its entirety. Suitable formatsinclude those shown in FIG. 2 of Brinkmann and Kontermann MAbs (2017)9(2): 182-212: antibody conjugates, e.g. IgG₂, F(ab′)₂ or CovX-Body: IgGor IgG-like molecules, e.g. IgG, chimeric IgG, κλ-body common HC; CH1/CLfusion proteins, e.g. scFv2-CH1/CL, VHH2-CH1/CL: ‘variable domain only’bispecific antigen-binding molecules, e.g. tandem scFv (taFV),triplebodies, diabodies (Db), dsDb, Db(kih), DART, scDB, dsFv-dsFv,tandAbs, triple heads, tandem dAb/VHH, tetravalent dAb.VHH; Non-Igfusion proteins, e.g. scFv₂-albumin, scDb-albumin, taFv-albumin,taFv-toxin, miniantibody, DNL-Fab₂, DNL-Fab₂-scFv,DNL-Fab₂-IgG-cytokine₂, ImmTAC (TCR-scFv); modified Fc and CH3 fusionproteins, e.g. scFv-Fc(kih), scFv-Fc(CH3 charge pairs), scFv-Fc(EW-RVT), scFv-fc (HA-TF), scFv-Fc (SEEDbody), taFv-Fc(kih),scFv-Fc(kih)-Fv, Fab-Fc(kih)-scFv, Fab-scFv-Fc(kih), Fab-scFv-Fc(BEAT),Fab-scFv-Fc (SEEDbody), DART-Fc, scFv-CH3(kih), TriFabs; Fc fusions,e.g. Di-diabody, scDb-Fc, taFv-Fc, scFv-Fc-scFv, HCAb-VHH, Fab-scFv-Fc,scFv₄-Ig, scFv2-Fcab; CH3 fusions, e.g. Dia-diabody, scDb-CH3; IgE/IgMCH2 fusions, e.g. scFv-EHD2-scFv, scFvMHD2-scFv; Fab fusion proteins,e.g. Fab-scFv (bibody), Fab-scFv2 (tribody), Fab-Fv, Fab-dsFv, Fab-VHH,orthogonal Fab-Fab; non-Ig fusion proteins, e.g. DNL-Fab₃,DNL-Fab₂-scFv, DNL-Fab₂-IgG-cytokine₂; asymmetric IgG or IgG-likemolecules, e.g. IgG(kih), IgG(kih) common LC, ZW1 IgG common LC,BicIonics common LC, CrossMab, CrossMab(kih), scFab-IgG(kih),Fab-scFab-IgG(kih), orthogonal Fab IgG(kih), DuetMab, CH3 chargepairs+CH1/CL charge pairs, hinge/CH3 charge pairs, SEED-body, Duobody,four-in-one-CrossMab(kih), LUZ-Y common LC; LUZ-Y scFab-IgG, FcFc*;appended and Fc-modified IgGs, e.g. IgG(kih)-Fv, IgG HA-TF-Fv,IgG(kih)scFab, scFab-Fc(kih)-scFv2, scFab-Fc(kih)-scFv, half DVD-Ig,DVI-Ig (four-in-one), CrossMab-Fab; modified Fc and CH3 fusion proteins,e.g. Fab-Fc(kih)-scFv, Fab-scFv-Fc(kih), Fab-scFv-Fc(BEAT),Fab-scFv-Fc-SEEDbody, TriFab; appended IgGs-HC fusions, e.g. IgG-HC,scFv, IgG-dAb, IgG-taFV, IgG-CrossFab, IgG-orthogonal Fab, IgG-(CαCβ)Fab, scFv-HC-IgG, tandem Fab-IgG (orthogonal Fab) Fab-IgG(CαCβ Fab),Fab-IgG(CR3), Fab-hinge-IgG(CR3); appended IgGs-LC fusions, e.g.IgG-scFv(LC), scFv(LC)-IgG, dAb-IgG; appended IgGs-HC and LC fusions,e.g. DVD-Ig, TVD-Ig, CODV-Ig, scFv₄-IgG, Zybody; Fc fusions, e.g.Fab-scFv-Fc, scFv₄-Ig; F(ab′)2 fusions, e.g. F(ab′)₂-scFv₂; CH1/CLfusion proteins e.g. scFv₂-CH1-hinge/CL; modified IgGs, e.g. DAF (two-inone-IgG), DutaMab, Mab²; and non-Ig fusions, e.g. DNL-Fab₄-IgG.

The skilled person is able to design and prepare bispecificantigen-binding molecules. Methods for producing bispecificantigen-binding molecules include chemically crosslinking ofantigen-binding molecules or antibody fragments, e.g. with reducibledisulphide or non-reducible thioether bonds, for example as described inSegal and Bast, 2001. Production of Bispecific Antigen-bindingmolecules. Current Protocols in Immunology. 14:IV:2.13:2.13.1-2.13.16,which is hereby incorporated by reference in its entirety. For example,N-succinimidyl-3-(−2-pyridyldithio)-propionate (SPDP) can be used tochemically crosslink e.g. Fab fragments via hinge region SH— groups, tocreate disulfide-linked bispecific F(ab)₂ heterodimers.

Other methods for producing bispecific antigen-binding molecules includefusing antibody-producing hybridomas e.g. with polyethylene glycol, toproduce a quadroma cell capable of secreting bispecific antibody, forexample as described in D. M. and Bast, B. J. 2001. Production ofBispecific Antigen-binding molecules. Current Protocols in Immunology.14:IV:2.13:2.13.1-2.13.16.

Bispecific antigen-binding molecules according to the present inventioncan also be produced recombinantly, by expression from e.g. a nucleicacid construct encoding polypeptides for the antigen-binding molecules,for example as described in Antibody Engineering: Methods and Protocols,Second Edition (Humana Press, 2012), at Chapter 40: Production ofBispecific Antigen-binding molecules: Diabodies and Tandem scFv (Hornigand Färber-Schwarz), or French, How to make bispecific antigen-bindingmolecules, Methods Mol. Med. 2000; 40:333-339, the entire contents ofboth of which are hereby incorporated by reference. For example, a DNAconstruct encoding the light and heavy chain variable domains for thetwo antigen-binding fragments (i.e. the light and heavy chain variabledomains for the antigen-binding fragment capable of binding HER3, andthe light and heavy chain variable domains for the antigen-bindingfragment capable of binding to another target protein), and includingsequences encoding a suitable linker or dimerization domain between theantigen-binding fragments can be prepared by molecular cloningtechniques. Recombinant bispecific antibody can thereafter be producedby expression (e.g. in vitro) of the construct in a suitable host cell(e.g. a mammalian host cell), and expressed recombinant bispecificantibody can then optionally be purified.

Fc Regions

In some embodiments the antigen-binding molecules of the presentinvention comprise an Fc region.

In IgG, IgA and IgD isotypes an Fc region is composed of CH2 and CH3regions from one polypeptide, and CH2 and CH3 regions from anotherpolypeptide. The CH2 and CH3 regions from the two polypeptides togetherform the Fc region. In IgM and IgE isotypes the Fc regions contain threeconstant domains (CH2, CH3 and CH4), and CH2 to CH4 from the twopolypeptides together form the Fc region.

In preferred embodiments in accordance with the various aspects of thepresent disclosure an Fc region comprises two polypeptides, eachpolypeptide comprising a CH2 region and a CH3 region.

In some embodiments, the antigen-binding molecule of the presentinvention comprises an Fc region comprising modification in one or moreof the CH2 and CH3 regions promoting association of the Fc region.Recombinant co-expression of constituent polypeptides of anantigen-binding molecule and subsequent association leads to severalpossible combinations. To improve the yield of the desired combinationsof polypeptides in antigen-binding molecules in recombinant production,it is advantageous to introduce in the Fc regions modification(s)promoting association of the desired combination of heavy chainpolypeptides. Modifications may promote e.g. hydrophobic and/orelectrostatic interaction between CH2 and/or CH3 regions of differentpolypeptide chains. Suitable modifications are described e.g. in Ha etal., Front. Immnol (2016) 7:394, which is hereby incorporated byreference in its entirety.

In some embodiments the antigen-binding molecule of the presentinvention comprises an Fc region comprising paired substitutions in theCH3 regions of the Fr region according to one of the following formats,as shown in Table 1 of Ha et al., Front. Immnol (2016) 7:394: KiH,KiH_(s-s), HA-TF, ZW1, 7.8.60 DD-KK, EW-RVT, EW-RVT_(s-s), SEED or A107.

In some embodiments, the Fc region comprises the “knob-into-hole” or“KiH” modification, e.g. as described e.g. in U.S. Pat. No. 7,695,936and Carter, J Immunol Meth 248, 7-15 (2001). In such embodiments, one ofthe CH3 regions of the Fc region comprises a “knob” modification, andthe other CH3 region comprises a “hole” modification. The “knob” and“hole” modifications are positioned within the respective CH3 regions sothat the “knob” can be positioned in the “hole” in order to promoteheterodimerisation (and inhibit homodimerisation) of the polypeptidesand/or stabilise heterodimers. Knobs are constructed by substitutingamino acids having small chains with those having larger side chains(e.g. tyrosine or tryptophan). Holes are created by substituting aminoacids having large side chains with those having smaller side chains(e.g. alanine or threonine).

In some embodiments, one of the CH3 regions of the Fc region of theantigen-binding molecule of the present invention comprises thesubstitution (numbering of positions/substitutions in the Fc, CH2 andCH3 regions herein is according to the EU numbering system as describedin Kabat et al., Sequences of Proteins of Immunological Interest, 5thEd. Public Health Service, National Institutes of Health, Bethesda, Md.,1991) T366W, and the other CH3 region of the Fc region comprises thesubstitution Y407V. In some embodiments, one of the CH3 regions of theFc region of the antigen-binding molecule comprises the substitutionT366W, and the other CH3 region of the Fc region comprises thesubstitutions T366S and L368A. In some embodiments, one of the CH3regions of the Fc region of the antigen-binding molecule comprises thesubstitution T366W, and the other CH3 region of the Fc region comprisesthe substitutions Y407V, T366S and L368A.

In some embodiments, the Fc region comprises the “DD-KK” modification asdescribed e.g. in WO 2014/131694 A1. In some embodiments, one of the CH3regions comprises the substitutions K392D and K409D, and the other CH3region of the Fc region comprises the substitutions E356K and D399K. Themodifications promote electrostatic interaction between the CH3 regions.

In some embodiments, the antigen-binding molecule of the presentinvention comprises an Fc region modified as described in Labrijn etal., Proc Natl Acad Sci USA. (2013) 110(13):5145-50, referred to as‘Duobody’ format. In some embodiments one of the CH3 regions comprisesthe substitution K409R, and the other CH3 region of the Fc regioncomprises the substitution K405L.

In some embodiments, the antigen-binding molecule of the presentinvention comprises an Fc region comprising the “EEE-RRR” modificationas described in Strop et al., J Mol Biol. (2012) 420(3):204-19. In someembodiments one of the CH3 regions comprises the substitutions D221E,P228E and L368E, and the other CH3 region of the Fc region comprises thesubstitutions D221R, P228R and K409R.

In some embodiments, the antigen-binding molecule comprises an Fc regioncomprising the “EW-RVT” modification described in Choi et al., MolCancer Ther (2013) 12(12):2748-59. In some embodiments one of the CH3regions comprises the substitutions K360E and K409W, and the other CH3region of the Fc region comprises the substitutions Q347R, D399V andF405T.

In some embodiments, one of the CH3 regions comprises the substitutionS354C, and the other CH3 region of the Fc region comprises thesubstitution Y349C. Introduction of these cysteine residues results information of a disulphide bridge between the two CH3 regions of the Fcregion, fruitier stabilizing the heterodimer (Carter (2001), J ImmunolMethods 248, 7-15).

In some embodiments, the Fc region comprises the “KiH_(s-s)”modification. In some embodiments one of the CH3 regions comprises thesubstitutions T366W and S354C, and the other CH3 region of the Fc regioncomprises the substitutions T366S, L368A, Y407V and Y349C.

In some embodiments, the antigen-binding molecule of the presentinvention comprises an Fc region comprising the “SEED” modification asdescribed in Davis et al., Protein Eng Des Sel (2010) 23(4):195-202, inwhich β-strand segments of human IgG1 CH3 and IgA CH3 are exchanged.

In some embodiments, one of the CH3 regions comprises the substitutionsS364H and F405A, and the other CH3 region of the Fc region comprises thesubstitutions Y349T and T394F (see e.g. Moore et al., MAbs (2011)3(6):546-57).

In some embodiments, one of the CH3 regions comprises the substitutionsT350V, L351Y, F405A and Y407V, and the other CH3 region of the Fc regioncomprises the substitutions T350V, T366L, K392L and T394W (see e.g. VonKreudenstein et al., MAbs (2013) 5(5):646-54).

In some embodiments, one of the CH3 regions comprises the substitutionsK360D, D399M and Y407A, and the other CH3 region of the Fc regioncomprises the substitutions E345R, Q347R, T366V and K409V (see e.g.Leaver-Fay et al., Structure (2016) 24(4):641-51).

In some embodiments, one of the CH3 regions comprises the substitutionsK370E and K409W, and the other CH3 region of the Fc region comprises thesubstitutions E357N, D399V and F405T (see e.g. Choi et al., PLoS One(2015) 10(12):e0145349).

Fc-mediated functions include Fc receptor binding, antibody-dependentcellular cytotoxicity (ADCC), antibody-dependent cell-mediatedphagocytosis (ADCP), complement-dependent cytotoxicity (CDC), formationof the membrane attack complex (MAC), cell degranulation, cytokineand/or chemokine production, and antigen processing and presentation.

Modifications to antibody Fc regions that influence Fc-mediatedfunctions are known in the art, such as those described e.g. in Wang etal., Protein Cell (2018) 9(1):63-73, which is hereby incorporated byreference in its entirety. Exemplary Fc region modifications known toinfluence antibody effector function are summarised in Table 1 of Wanget al., Protein Cell (2018) 9(1):63-73.

The combination of substitutions F243L/R292P/Y300L/V305I/P396L isdescribed in Stavenhagen et al. Cancer Res. (2007) to increase bindingto FcγRIIIa, and thereby enhance ADCC. The combination of substitutionsS239D/I332E or S239D/I332E/A330L is described in Lazar et al., Proc NatlAcad Sci USA. (2006) 103:4005-4010 to increase binding to FcγRIIIa, andthereby increase ADCC. The combination of substitutionsS239D/I332E/A330L is also described to decrease binding to FcγRIIb, andthereby increase ADCC. The combination of substitutionsS298A/E333A/K334A is described in Shields et al., J Biol Chem. (2001)276:6591-6604 to increase binding to FcγRIIIa, and thereby increaseADCC. The combination of substitutionsL234Y/L235Q/G236W/S239M/H268D/D270E/S298A in one heavy chain, and thecombination of substitutions D270E/K326D/A330M/K334E in the other heavychain, is described in Mimoto et al., MAbs. (2013): 5:229-236 toincrease binding to FcγRIIIa, and thereby increase ADCC. The combinationof substitutions G236A/S239D/I332E is described in Richards et al., MolCancer Ther. (2008) 7:2517-2527 to increase binding to FcγRIIa and toincrease binding to FcγRIIIa, and thereby increase ADCP.

The combination of substitutions K326W/E333S is described in Idusogie etal. J Immunol. (2001) 166(4):2571-5 to increase binding to C1q, andthereby increase CDC. The combination of substitutions S267E/H268F/S324Tis described in Moore et al. MAbs. (2010) 2(2):181-9 to increase bindingto C1q, and thereby increase CDC. The combination of substitutionsdescribed in Natsume et al., Cancer Res. (2008) 68(10):3863-72 isreported to increase binding to C1q, and thereby increase CDC. Thecombination of substitutions E345R/E430G/S440Y is described in Dieboideret al. Science (2014) 343(6176):1260-3 to increase hexamerisation, andthereby increase CDC.

The combination of substitutions M252Y/S254T/T256E is described inDall'Acqua et al. J Immunol. (2002) 169:5171-5180 to increase binding toFcRn at pH 6.0, and thereby increase antigen-binding molecule half-life.The combination of substitutions M428L/N434S is described in Zalevsky etal. Nat Biotechnol. (2010) 28:157-159 to increase binding to FcRn at pH6.0, and thereby increase antigen-binding molecule half-life.

Where a heavy chain constant region/Fc region/CH2-CH3 region/CH2region/CH3 region is described herein as comprisingposition(s)/substitution(s) “corresponding to” reference positions) subequivalent position(s)/substitution(s) in homologous heavy chainconstant regions/Fc regions/CH2-CH3 regions/CH2 regions/CH3 regions arecontemplated.

Where an Fc region is described as comprising specificposition(s)/substitution(s), the position(s)/substitution(s) may bepresent in one or both of the polypeptide chains which together form theFc region.

Unless otherwise specified, positions herein refer to positions of humanimmunoglobulin constant region amino acid sequences numbered accordingto the EU numbering system as described in Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md., 1991. By way ofillustration, the substitutions L242C and K334C in human IgG1 correspondto L>C substitution at position 125, and K>C substitution at position217 of the human IgG1 constant region numbered according to SEQ IDNO:171.

Homologous heavy chain constant regions are heavy chain constant regionscomprising an amino acid sequence having at least 60%, preferably one of70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or100% amino acid sequence identity to the heavy chain constant region ofHuman IgG1 (i.e. the amino acid sequence shown in SEQ ID NO:171).Homologous Fc regions are Fc regions comprised of polypeptidescomprising an amino acid sequence having at least 60%, preferably one of70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or100% amino acid sequence identity to CH2-CH3 region of Human IgG1 (i.e.the amino acid sequences shown in SEQ ID NO:174 and 175). Homologous CH2regions are CH2 regions comprising an amino acid sequence having atleast 60%, preferably one of 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to CH2region of Human IgG1 (i.e. the amino acid sequence shown in SEQ IDNO:174). Homologous CH3 regions are CH3 regions comprising an amino acidsequence having at least 60%, preferably one of 70%, 75%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequenceidentity to CH3 region of Human IgG1 (i.e. the amino acid sequence shownin SEQ ID NO:175).

Corresponding positions to those identified in human IgG1 can beidentified by sequence alignment which can be performed e.g. usingsequence alignment software such as ClustalOmega (Söding, J. 2005,Bioinformatics 21, 951-960).

In some embodiments the antigen-binding molecule of the presentinvention comprises an Fc region comprising modification to increase anFc-mediated function. In some embodiments the Fc region comprisesmodification to increase ADCC. In some embodiments the Fc regioncomprises modification to increase ADCP. In some embodiments the Fcregion comprises modification to increase CDC. An antigen-bindingmolecule comprising an Fc region comprising modification to increase anFc-mediated function (e.g. ADCC, ADCP, CDC) induces an increased levelof the relevant effector function as compared to an antigen-bindingmolecule comprising the corresponding unmodified Fc region.

In some embodiments, the antigen-binding molecule of the presentinvention comprises an Fc region comprising modification to increaseaffinity for one or more Fc receptors (e.g. FcγRIIa, FcγRIIIa).Modifications increasing affinity for Fc receptors can increaseFc-mediated effector function such as antibody-dependent cellularcytotoxicity (ADCC) and/or antibody-dependent cellular phagocytosis(ADCP). In some embodiments, the antigen-binding molecule of the presentinvention comprises an Fc region comprising modification to reduceaffinity for C1q; such modification reducing complement-dependentcytotoxicity (CDC), which can be desirable. In some embodiments, theantigen-binding molecule of the present invention comprises an Fc regioncomprising modification to increase hexamer formation. Modifications tothe Fc region capable of increasing affinity for one or more Fcreceptors, reducing affinity for C1q and/or increasing hexamer formationare described e.g. in Saxena and Wu Front Immunol. (2016) 7:580, whichis hereby incorporated by reference in its entirety. In some embodimentsthe antigen-binding molecule of the present invention comprises an Fcregion comprising CH2/CH3 comprising one or more of the substitutionsshown in Table 1 of Saxena and Wu Front Immunol. (2016) 7:580.

In some embodiments the antigen-binding molecule of the presentinvention comprises an Fc comprising modification to increase binding toan Fc receptor. In some embodiments the Fc region comprises modificationto increase binding to an Fcγ receptor. In some embodiments the Fcregion comprises modification to increase binding to one or more ofFcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa and FcγRIIIb. In someembodiments the Fc region comprises modification to increase binding toFcγRIIIa. In some embodiments the Fc region comprises modification toincrease binding to FcγRIIa. In some embodiments the Fc region comprisesmodification to increase binding to FcγRIIb. In some embodiments the Fcregion comprises modification to increase binding to FcRn. In someembodiments the Fc region comprises modification to increase binding toa complement protein. In some embodiments the Fc region comprisesmodification to increase or reduce binding to C1q. In some embodimentsthe Fc region comprises modification to promote hexamerisation of theantigen-binding molecule. In some embodiments the Fc region comprisesmodification to increase antigen-binding molecule half-life. In someembodiments the Fc region comprises modification to increaseco-engagement.

In this specification an “Fcγ receptor” may be from any species, andincludes isoforms, fragments, variants (including mutants) or homologuesfrom any species. Similarly, “FcγRI”, “FcγRIIa”, “FcγRIIb”, “FcγRIIc”,“FcγRIIIa” and “FcγRIIIb” refer respectively toFcγRI/FcγRIIa/FcγRIIb/FcγRIIc/FcγRIIIa/FcγRIIIb from any species, andinclude isoforms, fragments, variants (including mutants) or homologuesfrom any species. Humans have six different classes of Fcγ receptor(mouse orthologues are shown in brackets): FcγRI (mFcγRI), FcγRIIa(mFcγRIII), FcγRIIb (mFcγRIIb), FcγRIIc, FcγRIIIa (mFcγRIV) andFcγRIIIb. Variant Fcγ receptors include e.g. the 158V and 158Fpolymorphs of human FcγRIIIa, and the 167H and 167R polymorphs of humanFcγRIIa.

In some embodiments the antigen-binding molecule of the presentinvention comprises an Fc region comprising (e.g. comprising one morepolypeptides comprising a heavy chain constant region, or a CH2-CH3region, comprising) one or more (e.g. 1, 2, 3, 4, 5, 6, 7 or 8) of thefollowing: C at the position corresponding to position 242; C at theposition corresponding to position 334; A at the position correspondingto position 236; D at the position corresponding to position 239; E atthe position corresponding to position 332; L at the positioncorresponding to position 330; K at the position corresponding toposition 345; and G at the position corresponding to position 430.

In some embodiments the antigen-binding molecule of the presentinvention comprises an Fc region comprising (e.g. comprising one morepolypeptides comprising a heavy chain constant region, or a CH2-CH3region, comprising) one or more (e.g. 1, 2, 3, 4, 5, 6, 7 or 8) of thefollowing substitutions (or corresponding substitutions): L242C, K334C,G236A, S239D, I332E, A330L, E345K, and E430G.

In some embodiments the antigen-binding molecule comprises an Fc regioncomprising (e.g. comprising one more polypeptides comprising a heavychain constant region, a CH2-CH3 region, or a CH2 region, comprising) aC at the position corresponding to position 242. In some embodiments theFc region comprises (e.g. comprises one more polypeptides comprising aheavy chain constant region, a CH2-CH3 region, or a CH2 region,comprising) a C at the position corresponding to position 334. In someembodiments the Fc region comprises (e.g. comprises one morepolypeptides comprising a heavy chain constant region, a CH2-CH3 region,or a CH2 region, comprising) a C at the position corresponding toposition 242 and a C at the position corresponding to position 334.

In some embodiments the antigen-binding molecule comprises an Fc regioncomprising (e.g. comprising one more polypeptides comprising a heavychain constant region, a CH2-GFH region, or a CH2 region, comprising) anA at the position corresponding to position 236. In some embodiments theFc region comprises (e.g. comprises one more polypeptides comprising aheavy chain constant region, a CH2-CH3 region, or a CH2 region,comprising) a D at the position corresponding to position 239. In someembodiments the Fc region comprises (e.g. comprises one morepolypeptides comprising a heavy chain constant region, a CH2-CH3 region,or a CH2 region, comprising) an A at the position corresponding toposition 236, and a D at the position corresponding to position 239.

In some embodiments the antigen-binding molecule comprises an Fc regioncomprising (e.g. comprising one more polypeptides comprising a heavychain constant region, a CH2-CH3 region, or a CH2 region, comprising) anE at the position corresponding to position 332. In some embodiments theFc region comprises (e.g. comprises one more polypeptides comprising aheavy chain constant region, a CH2-CH3 region, or a CH2 region,comprising) an A at the position corresponding to position 236, a D atthe position corresponding to position 239, and an E at the positioncorresponding to position 332.

In some embodiments the antigen-binding molecule comprises an Fc regioncomprising (e.g. comprising one more polypeptides comprising a heavychain constant region, a CH2-CH3 region, or a CH2 region, comprising) anL at the position corresponding to position 330. In some embodiments theFc region comprises (e.g. comprises one more polypeptides comprising aheavy chain constant region, a CH2-CH3 region, or a CH2 region,comprising) an A at the position corresponding to position 236, a D atthe position corresponding to position 239, an E at the positioncorresponding to position 332, and an L at the position corresponding toposition 330.

In some embodiments the antigen-binding molecule comprises an Fc regioncomprising (e.g. comprising one more polypeptides comprising a heavychain constant region, a CH2-CH3 region, or a CH3 region, comprising) aK at the position corresponding to position 345. In some embodiments theFc region comprises (e.g. comprises one more polypeptides comprising aheavy chain constant region, a CH2-CH3 region, or a CH3 region,comprising) a G at the position corresponding to position 430. In someembodiments the Fc region comprises (e.g. comprises one morepolypeptides comprising a heavy chain constant region, a CH2-CH3 region,or a CH2 region, comprising) a K at the position corresponding toposition 345, and a G at the position corresponding to position 430.

In some embodiments the antigen-binding molecule comprises an Fc regioncomprising (e.g. comprising one more polypeptides comprising a heavychain constant region, a CH2-CH3 region, or a CH2 region, comprising) aC at the position corresponding to position 242, a C at the positioncorresponding to position 334, an A at the position corresponding toposition 236, and a D at the position corresponding to position 239.

In some embodiments the antigen-binding molecule comprises an Fc regioncomprising (e.g. comprising one more polypeptides comprising a heavychain constant region, a CH2-CH3 region, or a CH2 region, comprising) aC at the position corresponding to position 242, a C at the positioncorresponding to position 334, an A at the position corresponding toposition 236, a D at the position corresponding to position 239, and anE at the position corresponding to position 332.

In some embodiments the antigen-binding molecule comprises an Fc regioncomprising (e.g. comprising one more polypeptides comprising a heavychain constant region, a CH2-CH3 region, or a CH2 region, comprising) aC at the position corresponding to position 242, a C at the positioncorresponding to position 334, an A at the position corresponding toposition 236, a D at the position corresponding to position 239, an E atthe position corresponding to position 332, and an L at the positioncorresponding to position 330.

In some embodiments the antigen-binding molecule comprises an Fc regioncomprising (e.g. comprising one more polypeptides comprising a heavychain constant region, or a CH2-CH3 region, comprising) a C at theposition corresponding to position 242, a C at the positioncorresponding to position 334, a K at the position corresponding toposition 345, and a G at the position corresponding to position 430.

In some embodiments the antigen-binding molecule comprises an Fc regioncomprising (e.g. comprising one more polypeptides comprising a heavychain constant region, a CH2-CH3 region, or a CH2 region, comprising)the substitution L242C (or an equivalent substitution). In someembodiments the Fc region comprises (e.g. comprises one morepolypeptides comprising a heavy chain constant region, a CH2-CH3 region,or a CH2 region, comprising) the substitution K334C (or an equivalentsubstitution). In some embodiments the Fc region comprises (e.g,comprises one more polypeptides comprising a heavy chain constantregion, a CH2-CH3 region, or a CH2 region, comprising) the substitutionL242C (or an equivalent substitution) and the substitution K334C (or anequivalent substitution).

In some embodiments the antigen-binding molecule comprises an Fc regioncomprising (e.g. comprising one more polypeptides comprising a heavychain constant region, a CH2-CH3 region, or a CH2 region, comprising)the substitution G236A (or an equivalent substitution). In someembodiments the Fc region comprises (e.g. comprises one morepolypeptides comprising a heavy chain constant region, a CH2-CH3 region,or a CH2 region, comprising) the substitution S239D (or an equivalentsubstitution). In some embodiments the Fc region comprises (e.g.comprises one more polypeptides comprising a heavy chain constantregion, a CH2-CH3 region, or a CH2 region, comprising) the substitutionG236A (or an equivalent substitution), and the substitution S239D (or anequivalent substitution).

In some embodiments the antigen-binding molecule comprises an Fc regioncomprising (e.g. comprising one more polypeptides comprising a heavychain constant region, a CH2-CH3 region, or a CH2 region, comprising)the substitution I332E (or an equivalent substitution). In someembodiments the Fc region comprises (e.g. comprises one morepolypeptides comprising a heavy chain constant region, a CH2-CH3 region,or a CH2 region, comprising) the substitution G236A (or an equivalentsubstitution), the substitution S239D (or an equivalent substitution),and the substitution I332E (or an equivalent substitution).

In some embodiments the antigen-binding molecule comprises an Fc regioncomprising (e.g, comprising one more polypeptides comprising a heavychain constant region, a CH2-CH3 region, or a CH2 region, comprising)the substitution A330L (or an equivalent substitution). In someembodiments the Fc region comprises (e.g. comprises one morepolypeptides comprising a heavy chain constant region, a CH2-CH3 region,or a CH2 region, comprising) the substitution G236A (or an equivalentsubstitution), the substitution S239D (or an equivalent substitution),the substitution I332E (or an equivalent substitution), and thesubstitution A330L (or an equivalent substitution).

In some embodiments the antigen-binding molecule comprises an Fc regioncomprising (e.g. comprising one more polypeptides comprising a heavychain constant region, a CH2-CH3 region, or a CH3 region, comprising)the substitution E345K (or an equivalent substitution). In someembodiments the Fc region comprises (e.g. comprises one morepolypeptides comprising a heavy chain constant region, a CH2-CH3 region,or a CH3 region, comprising) the substitution E430G (or an equivalentsubstitution). In some embodiments the Fc region comprises (e.g.comprises one more polypeptides comprising a heavy chain constantregion, a CH2-CH3 region, or a CH2 region, comprising) the substitutionE345K (or an equivalent substitution), and the substitution E430G (or anequivalent substitution).

In some embodiments the antigen-binding molecule comprises an Fc regioncomprising (e.g. comprising one more polypeptides comprising a heavychain constant region, a CH2-CH3 region, or a CH2 region, comprising)the substitution L242C (or an equivalent substitution), the substitutionK334C (or an equivalent substitution), the substitution G236A (or anequivalent substitution), and the substitution S239D (or an equivalentsubstitution).

In some embodiments the antigen-binding molecule comprises an Fc regioncomprising (e.g. comprising one more polypeptides comprising a heavychain constant region, a CH2-CH3 region, or a CH2 region, comprising)the substitution L242C (or an equivalent substitution), the substitutionK334C (or an equivalent substitution), the substitution G236A (or anequivalent substitution), the substitution S239D (or an equivalentsubstitution), and the substitution I332E (or an equivalentsubstitution).

In some embodiments the antigen-binding molecule comprises an Fc regioncomprising (e.g. comprising one more polypeptides comprising a heavychain constant region, a CH2-CH3 region, or a CH2 region, comprising)the substitution L242C (or an equivalent substitution), the substitutionK334C (or an equivalent substitution), the substitution G236A (or anequivalent substitution), the substitution S239D (or an equivalentsubstitution), the substitution I332E (or an equivalent substitution),and the substitution A330L (or an equivalent substitution).

In some embodiments the antigen-binding molecule comprises an Fc regioncomprising (e.g. comprising one more polypeptides comprising a heavychain constant region, or a CH2-CH3 region, comprising) the substitutionL242C (or an equivalent substitution), the substitution K334C (or anequivalent substitution), the substitution E345K (or an equivalentsubstitution), and the substitution E430G (or an equivalentsubstitution).

In some embodiments the antigen-binding molecule comprises an Fc regioncomprising (e.g. comprising one more polypeptides comprising a heavychain constant region, or a CH2-CH3 region, comprising) one or more(e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12) of the following: L atthe position corresponding to position 243, P at the positioncorresponding to position 292, L at the position corresponding toposition 300, 1 at the position corresponding to position 305 and L atthe position corresponding to position 396; at the positioncorresponding to position 239 and E at the position corresponding toposition 332; 0 at the position corresponding to position 239, E at theposition corresponding to position 332 and L at the positioncorresponding to position 330; A at the position corresponding toposition 298, A at the position corresponding to position 333 and A atthe position corresponding to position 334; Y at the positioncorresponding to position 234, Q at the position corresponding toposition 235, W at the position corresponding to position 236, M at theposition corresponding to position 239, D at the position correspondingto position 268, E at the position corresponding to position 270 and Aat the position corresponding to position 298; E at the positioncorresponding to position 270, D at the position corresponding toposition 326, M at the position corresponding to position 330 and E atthe position corresponding to position 334; A at the positioncorresponding to position 236, D at the position corresponding toposition 239 and E at the position corresponding to position 332; W atthe position corresponding to position 326 and S at the positioncorresponding to position 333; E at the position corresponding toposition 267, F at the position corresponding to position 268 and T atthe position corresponding to position 324; R at the positioncorresponding to position 345, G at the position corresponding toposition 430 and Y at the position corresponding to position 440; Y atthe position corresponding to position 252, T at the positioncorresponding to position 254 and E at the position corresponding toposition 256, and L at the position corresponding to position 428 and Sat the position corresponding to position 434.

In some embodiments the antigen-binding molecule comprises an Fc regioncomprising (e.g. comprising one more polypeptides comprising a heavychain constant region, or a CH2-CH3 region, comprising) one or more(e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12) of the followingcombinations of substitutions (or corresponding substitutions):F243L/R292P/Y300L/V305I/P396L; S239D/I332E; S239D/I332E/A330L;S298A/E333A/K334A; L234Y/L235Q/G236W/S239M/H268D/D270E/S298A;D270E/K326D/A330M/K334E; G238A/S239D/I332E; K326W/E333S;S287E/H288F/S324T; E345R/E430G/S440Y; M252Y/S254T/T256E; andM428L/N434S.

Polypeptides

The present invention also provides polypeptide constituents ofantigen-binding molecules. The polypeptides may be provided in isolatedor substantially purified form.

The antigen-binding molecule of the present invention may be, or maycomprise, a complex of polypeptides.

In the present specification where a polypeptide comprises more than onedomain or region, it will be appreciated that the plural domains/regionsare preferably present in the same polypeptide chain. That is, thepolypeptide comprises more than one domain or region is a fusionpolypeptide comprising the domains/regions.

In some embodiments a polypeptide according to the present inventioncomprises, or consists of, a VH as described herein. In some embodimentsa polypeptide according to the present invention comprises, or consistsof, a VL as described herein.

In some embodiments, the polypeptide additionally comprises one or moreantibody heavy chain constant regions (CH). In some embodiments, thepolypeptide additionally comprises one or more antibody light chainconstant regions (CL). In some embodiments, the polypeptide comprises aCH1, CH2 region and/or a CH3 region of an immunoglobulin (Ig).

In some embodiments the polypeptide comprises one or more regions of animmunoglobulin heavy chain constant sequence. In some embodiments thepolypeptide comprises a CH1 region as described herein. In someembodiments the polypeptide comprises a CH1-CH2 hinge region asdescribed herein. In some embodiments the polypeptide comprises a CH2region as described herein. In some embodiments the polypeptidecomprises a CH3 region as described herein. In some embodiments thepolypeptide comprises a CH2-CH3 region as described herein.

In some embodiments the polypeptide comprises a CH3 region comprisingany one of the following amino acid substitutions/combinations of aminoacid substitutions (shown e.g. in Table 1 of Ha et al., Front. Immnol(2016) 7:394, incorporated by reference hereinabove): T366W; T366S,L368A and Y407V; T366W and S354C; T366S, L368A, Y407V and Y349C; S364Hand F405A; Y349T and T394F, T350V, L351Y, F405A and Y407V; T350V, T366L,K392L and T394W: K360D, D399M and Y407A; E345R, Q347R, T366V and K409V;K409D and K392D; D399K and E356K; K360E and K409W; Q347R, D399V andF405T; K360E, K409W and Y349C; Q347R, D399V, F405T and S354C; K370E andK409W, and E357N, D399V and F405T.

In some embodiments the CH2 and/or CH3 regions of the polypeptidecomprise one or more amino acid substitutions for promoting associationof the polypeptide with another polypeptide comprising a CH2 and/or CH3region.

In some embodiments the polypeptide comprises one or more regions of animmunoglobulin light chain constant sequence. In some embodiments thepolypeptide comprises a CL region as described herein.

In some embodiments, the polypeptide according to the present inventioncomprises a structure from N- to C-terminus according to one of thefollowing:

-   -   (i) VH    -   (ii) VL    -   (iii) VH-CH1    -   (iv) VL-CL    -   (v) VL-CH1    -   (vi) VH-CL    -   (vii) VH-CH1-CH2-CH3    -   (viii) VL-CL-CH2-CH3    -   (ix) VL-CH1-CH2-CH3    -   (x) VH-CL-CH2-CH3

Also provided by the present invention are antigen-binding moleculescomposed of the polypeptides of the present invention. In someembodiments, the antigen-binding molecule of the present inventioncomprises one of the following combinations of polypeptides:

-   -   (A) VH+VL    -   (B) VH-CH1+VL-CL    -   (C) VL-CH1+VH-CL    -   (D) VH-CH1-CH2-CH3+VL-CL    -   (E) VH-CL-CH2-CH3+VL-CH1    -   (F) VL-CH1-CH2-CH3+VH-CL    -   (G) VL-CL-CH2-CH3+VH-CH1    -   (H) VH-CH1-CH2-CH3+VL-CL-CH2-CH3    -   (I) VH-CL-CH2-CH3+VL-CH1-CH2-CH3

In some embodiments the antigen-binding molecule comprises more than oneof a polypeptide of the combinations shown in (A) to (I) above. By wayof example, with reference to (D) above, in some embodiments theantigen-binding molecule comprises two polypeptides comprising thestructure VH-CH1-CH2-CH3, and two polypeptides comprising the structureVL-CL.

In some embodiments, the antigen-binding molecule of the presentinvention comprises one of the following combinations of polypeptides:

-   -   (J) VH (anti-HER3)+VL (anti-HER3)    -   (K) VH (anti-HER3)-CH1+VL (anti-HER3)-CL    -   (L) VL (anti-HER3)-CH1+VH (anti-HER3)-CL    -   (M) VH (anti-HER3)-CH1-CH2-CH3+VL (anti-HER3)-CL    -   (N) VH (anti-HER3)-CL-CH2-CH3+VL (anti-HER3)-CH1    -   (O) VL (anti-HER3)-CH1-CH2-CH3+VH (anti-HER3)-CL    -   (P) VL (anti-HER3)-CL-CH2-CH3+VH (anti-HER3)-CH1    -   (Q) VH (anti-HER3)-CH1-CH2-CH3+VL (anti-HER3)-CL-CH2-CH3    -   (R) VH (anti-HER3)-CL-CH2-CH3+VL (anti-HER3)-CH1-CH2-CH3

Wherein: “VH(anti-HER3)” refers to the VH of an antigen-binding moleculecapable of binding to HER3 as described herein, e.g. as defined in oneof (1) to (61) above; “VL(anti-HER3)” refers to the VL of anantigen-binding molecule capable of binding to HER3 as described herein,e.g. as defined in one of (62) to (119) above.

In some embodiments the polypeptide comprises or consists of an aminoacid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequenceidentity to the amino acid sequence of one of SEQ ID NOs:187 to 223.

Linkers and Additional Sequences

In some embodiments the antigen-binding molecules and polypeptides ofthe present invention comprise a hinge region. In some embodiments ahinge region is provided between a CH1 region and a CH2 region. In someembodiments a hinge region is provided between a CL region and a CH2region. In some embodiments the hinge region comprises, or consists of,an amino acid sequence having at least 70%, preferably one of 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acidsequence identity to the amino acid sequence of SEQ ID NO:173.

In some embodiments the antigen-binding molecules and polypeptides ofthe present invention comprise one or more linker sequences betweenamino acid sequences. A linker sequence may be provided at one or bothends of one or more of a VH, VL, CH1-CH2 hinge region, CH2 region and aCH3 region of the antigen-binding molecule/polypeptide.

Linker sequences are known to the skilled person, and are described, forexample in Chen et al., Adv Drug Deliv Rev (2013) 65(10): 1357-136$,which is hereby incorporated by reference in its entirety. In someembodiments, a linker sequence may be a flexible linker sequence.Flexible linker sequences allow for relative movement of the amino acidsequences which are linked by the linker sequence. Flexible linkers areknown to the skilled person, and several are identified in Chen et al.,Adv Drug Deliv Rev (2013) 65(10): 1357-1369. Flexible linker sequencesoften comprise high proportions of glycine and/or serine residues.

In some embodiments, the linker sequence comprises at least one glycineresidue and/or at least one serine residue. In some embodiments thelinker sequence consists of glycine and serine residues. In someembodiments, the linker sequence has a length of 1-2, 1-3, 1-4, 1-5 or1-10 amino acids.

The antigen-binding molecules and polypeptides of the present inventionmay additionally comprise further amino acids or sequences of aminoacids. For example, the antigen-binding molecules and polypeptides maycomprise amino acid sequence(s) to facilitate expression, folding,trafficking, processing, purification or detection of theantigen-binding molecule/polypeptide. For example, the antigen-bindingmolecule/polypeptide may comprise a sequence encoding a His, (e.g.6×His), Myc, GST, MBP, FLAG, HA, E, or Biotin tag, optionally at the N-or C-terminus of the antigen-binding molecule/polypeptide. In someembodiments the antigen-binding molecule/polypeptide comprises adetectable moiety, e.g. a fluorescent, luminescent, immuno-detectable,radio, chemical, nucleic acid or enzymatic label.

The antigen-binding molecules and polypeptides of the present inventionmay additionally comprise a signal peptide (also known as a leadersequence or signal sequence). Signal peptides normally consist of asequence of 5-30 hydrophobic amino acids, which form a single alphahelix. Secreted proteins and proteins expressed at the cell surfaceoften comprise signal peptides.

The signal peptide may be present at the N-terminus of theantigen-binding molecule/polypeptide, and may be present in the newlysynthesised antigen-binding molecule/polypeptide. The signal peptideprovides for efficient trafficking and secretion of the antigen-bindingmolecule/polypeptide. Signal peptides are often removed by cleavage, andthus are not comprised in the mature antigen-bindingmolecule/polypeptide secreted from the cell expressing theantigen-binding molecule/polypeptide.

Signal peptides are known for many proteins, and are recorded indatabases such as GenBank, UniProt, Swiss-Prot, TrEMBL, ProteinInformation Resource, Protein Data Bank, Ensembl, and InterPro, and/orcan be identified/predicted e.g. using amino acid sequence analysistools such as SignalP (Petersen et al., 2011 Nature Methods 8: 785-786)or Signal-BLAST (Frank and Sippl, 2008 Bioinformatics 24: 2172-2176).

In some embodiments, the signal peptide of the antigen-bindingmolecule/polypeptide of the present invention comprises, or consists of,an amino acid sequence having at least 80%, 85% 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identityto the amino acid sequence of one of SEQ ID NOs:178 to 186.

Labels and Conjugates

In some embodiments the antigen-binding molecules of the presentinvention additionally comprise a detectable moiety.

In some embodiments the antigen-binding molecule comprises a detectablemoiety, e.g. a fluorescent label, phosphorescent label, luminescentlabel, immuno-detectable label (e.g. an epitope tag), radiolabel,chemical, nucleic acid or enzymatic label. The antigen-binding moleculemay be covalently or non-covalently labelled with the detectable moiety.

Fluorescent labels include e.g. fluorescein, rhodamine, allophycocyanin,eosine and NDB, green fluorescent protein (GFP) chelates of rare earthssuch as europium (Eu), terbium (Tb) and samarium (Sm), tetramethylrhodamine, Texas Red, 4-methyl umbelliferone, 7-amino-4-methyl coumarin,Cy3, and Cy5. Radiolabels include radioisotopes such as Iodine¹²³,Iodine¹²⁵, Iodine¹²⁶, Iodine¹³¹, Iodine¹³³, Bromine⁷⁷, Technetium^(99m),Indium¹¹¹, Indium^(113m), Gallium⁶⁷, Gallium⁶⁸, Ruthenium⁹⁵,Ruthenium⁹⁷, Ruthenium¹⁰³, Ruthenium¹⁰⁵, Mercury²⁰⁷, Mercury²⁰³,Rhenium^(99m), Rhenium¹⁰¹, Rhenium¹⁰⁵, Scandium⁴⁷, Tellurium^(121m),Tellurium^(122m), Tellurium^(125m), Thulium¹⁶⁵, Thulium¹⁶⁷, Thulium¹⁶⁸,Copper⁶⁷, Fluorine¹⁸, Yttrium⁹⁰, Palladium¹⁰⁰, Bismuth²¹⁷ andAntimony²¹¹. Luminescent labels include as radioluminescent,chemiluminescent (e.g. acridinium ester, luminal, isoluminol) andbioluminescent labels. Immuno-detectable labels include haptens,peptides/polypeptides, antibodies, receptors and ligands such as biotin,avidin, streptavidin or digoxigenin. Nucleic acid labels includeaptamers. Enzymatic labels include e.g. peroxidase, alkalinephosphatase, glucose oxidase, beta-galactosidase and luciferase.

In some embodiments the antigen-binding molecules of the presentinvention are conjugated to a chemical moiety. The chemical moiety maybe a moiety for providing a therapeutic effect. Antibody-drug conjugatesare reviewed e.g. in Parslow et al., Biomedicines. 2016 September;4(3):14. In some embodiments, the chemical moiety may be a drug moiety(e.g. a cytotoxic agent). In some embodiments, the drug moiety may be achemotherapeutic agent. In some embodiments, the drug moiety is selectedfrom calicheamicin, DM1, DM4, monomethylauristatin E (MMAE),monomethylauristatin F (MMAF), SN-38, doxorubicin, duocarmycin, D6.5 andPBD.

Particular Exemplary Embodiments of the Antigen-Binding Molecules

In some embodiments the antigen-binding molecule comprises, or consistsof:

-   -   (i) two polypeptides comprising, or consisting of, an amino acid        sequence having at least 70%, preferably one of 75%, 80%, 85%,        90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino        acid sequence identity to the amino acid sequence of NO:187, and    -   (ii) two polypeptides comprising, or consisting of, an amino        acid sequence having at least 70%, preferably one of 75%, 80%,        85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%        amino acid sequence identity to the amino acid sequence of        NO:188.

In some embodiments the antigen-binding molecule comprises, or consistsof:

-   -   (i) two polypeptides comprising, or consisting of, an amino acid        sequence having at least 70%, preferably one of 75%, 80%, 85%,        90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino        acid sequence identity to the amino acid sequence of NO:189; and    -   (ii) two polypeptides comprising, or consisting of, an amino        acid sequence having at least 70%, preferably one of 75%, 80%,        85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%        amino acid sequence identity to the amino acid sequence of        NO:190.

In some embodiments the antigen-binding molecule comprises, or consistsof:

-   -   (i) two polypeptides comprising, or consisting of, an amino acid        sequence having at least 70%, preferably one of 75%, 80%, 85%,        90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino        acid sequence identity to the amino acid sequence of NO:191; and    -   (ii) two polypeptides comprising, or consisting of, an amino        acid sequence having at least 70%, preferably one of 75%, 80%,        85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%        amino acid sequence identity to the amino acid sequence of        NO:192.

In some embodiments the antigen-binding molecule comprises, or consistsof:

-   -   (i) two polypeptides comprising, or consisting of, an amino acid        sequence having at least 70%, preferably one of 75%, 80%, 85%,        90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino        acid sequence identity to the amino acid sequence of NO:193; and    -   (ii) two polypeptides comprising, or consisting of, an amino        acid sequence having at least 70%, preferably one of 75%, 80%,        85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%        amino acid sequence identity to the amino acid sequence of        NO:195.

In some embodiments the antigen-binding molecule comprises, or consistsof:

-   -   (i) two polypeptides comprising, or consisting of, an amino acid        sequence having at least 70%, preferably one of 75%, 80%, 85%,        90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino        acid sequence identity to the amino acid sequence of NO:194; and    -   (ii) two polypeptides comprising, or consisting of, an amino        acid sequence having at least 70%, preferably one of 75%, 80%,        85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%        amino acid sequence identity to the amino acid sequence of        NO:195.

In some embodiments the antigen-binding molecule comprises, or consistsof:

-   -   (i) two polypeptides comprising, or consisting of, an amino acid        sequence having at least 70%, preferably one of 75%, 80%, 85%,        90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino        acid sequence identity to the amino acid sequence of NO:196; and    -   (ii) two polypeptides comprising, or consisting of, an amino        acid sequence having at least 70%, preferably one of 75%, 80%,        85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%        amino acid sequence identity to the amino acid sequence of        NO:195.

In some embodiments the antigen-binding molecule comprises, or consistsof:

-   -   (i) two polypeptides comprising, or consisting of, an amino acid        sequence having at least 70%, preferably one of 75%, 80%, 85%,        90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino        acid sequence identity to the amino acid sequence of NO:197; and    -   (ii) two polypeptides comprising, or consisting of, an amino        acid sequence having at least 70%, preferably one of 75%, 80%,        85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%        amino acid sequence identity to the amino acid sequence of        NO:199.

In some embodiments the antigen-binding molecule comprises, or consistsof:

-   -   (i) two polypeptides comprising, or consisting of, an amino acid        sequence having at least 70%, preferably one of 75%, 80%, 85%,        90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino        acid sequence identity to the amino acid sequence of NO:198; and    -   (ii) two polypeptides comprising, or consisting of, an amino        acid sequence having at least 70%, preferably one of 75%, 80%,        85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%        amino acid sequence identity to the amino acid sequence of        NO:199.

In some embodiments the antigen-binding molecule comprises, or consistsof:

-   -   (i) two polypeptides comprising, or consisting of, an amino acid        sequence having at least 70%, preferably one of 75%, 80%, 85%,        90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino        acid sequence identity to the amino acid sequence of NO:200; and    -   (ii) two polypeptides comprising, or consisting of, an amino        acid sequence having at least 70%, preferably one of 75%, 80%,        85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%        amino acid sequence identity to the amino acid sequence of        NO:201.

In some embodiments the antigen-binding molecule comprises, or consistsof:

-   -   (i) two polypeptides comprising, or consisting of, an amino acid        sequence having at least 70%, preferably one of 75%, 80%, 85%,        90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino        acid sequence identity to the amino acid sequence of NO:202; and    -   (ii) two polypeptides comprising, or consisting of, an amino        acid sequence having at least 70%, preferably one of 75%, 80%,        85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%        amino acid sequence identity to the amino acid sequence of        NO:203.

In some embodiments the antigen-binding molecule comprises, or consistsof:

-   -   (i) two polypeptides comprising, or consisting of, an amino acid        sequence having at least 70%, preferably one of 75%, 80%, 85%,        90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino        acid sequence identity to the amino acid sequence of NO:204, and    -   (ii) two polypeptides comprising, or consisting of, an amino        acid sequence having at least 70%, preferably one of 75%, 80%,        85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%        amino acid sequence identity to the amino acid sequence of        NO:205.

In some embodiments the antigen-binding molecule comprises, or consistsof:

-   -   (i) two polypeptides comprising, or consisting of, an amino acid        sequence having at least 70%, preferably one of 75%, 80%, 85%,        90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino        acid sequence identity to the amino acid sequence of NO:206; and    -   (ii) two polypeptides comprising, or consisting of, an amino        acid sequence having at least 70%, preferably one of 75%, 80%,        85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%        amino acid sequence identity to the amino acid sequence of        NO:207.

In some embodiments the antigen-binding molecule comprises, or consistsof:

-   -   (i) two polypeptides comprising, or consisting of, an amino acid        sequence having at least 70%, preferably one of 75%, 80%, 85%,        90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino        acid sequence identity to the amino acid sequence of NO:208; and    -   (ii) two polypeptides comprising, or consisting of, an amino        acid sequence having at least 70%, preferably one of 75%, 80%,        85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%        amino acid sequence identity to the amino acid sequence of        NO:209.

In some embodiments the antigen-binding molecule comprises, or consistsof:

-   -   (i) two polypeptides comprising, or consisting of, an amino acid        sequence having at least 70%, preferably one of 75%, 80%, 85%,        90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino        acid sequence identity to the amino acid sequence of NO:210; and    -   (ii) two polypeptides comprising, or consisting of, an amino        acid sequence having at least 70%, preferably one of 75%, 80%,        85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%        amino acid sequence identity to the amino acid sequence of        NO:211.

In some embodiments the antigen-binding molecule comprises, or consistsof:

-   -   (i) two polypeptides comprising, or consisting of, an amino acid        sequence having at least 70%, preferably one of 75%, 80%, 85%,        90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino        acid sequence identity to the amino acid sequence of NO:212; and    -   (ii) two polypeptides comprising, or consisting of, an amino        acid sequence having at least 70%, preferably one of 75%, 80%,        85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%        amino acid sequence identity to the amino acid sequence of        NO:213.

In some embodiments the antigen-binding molecule comprises, or consistsof:

-   -   (i) two polypeptides comprising, or consisting of, an amino acid        sequence having at least 70%, preferably one of 75%, 80%, 85%,        90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino        acid sequence identity to the amino acid sequence of NO:214, and    -   (ii) two polypeptides comprising, or consisting of, an amino        acid sequence having at least 70%, preferably one of 75%, 80%,        85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%        amino acid sequence identity to the amino acid sequence of        NO:215.

In some embodiments the antigen-binding molecule comprises, or consistsof:

-   -   (i) two polypeptides comprising, or consisting of, an amino acid        sequence having at least 70%, preferably one of 75%, 80%, 85%,        90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino        acid sequence identity to the amino acid sequence of NO:216; and    -   (ii) two polypeptides comprising, or consisting of, an amino        acid sequence having at least 70%, preferably one of 75%, 80%,        85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%        amino acid sequence identity to the amino acid sequence of        NO:217.

In some embodiments the antigen-binding molecule comprises, or consistsof:

-   -   (i) two polypeptides comprising, or consisting of, an amino acid        sequence having at least 70%, preferably one of 75%, 80%, 85%,        90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino        acid sequence identity to the amino acid sequence of NO:218; and    -   (ii) two polypeptides comprising, or consisting of, an amino        acid sequence having at least 70%, preferably one of 75%, 80%,        85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%        amino acid sequence identity to the amino acid sequence of        NO:219.

In some embodiments the antigen-binding molecule comprises, or consistsof:

-   -   (i) two polypeptides comprising, or consisting of, an amino acid        sequence having at least 70%, preferably one of 75%, 80%, 85%,        90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino        acid sequence identity to the amino acid sequence of NO:220; and    -   (ii) two polypeptides comprising, or consisting of, an amino        acid sequence having at least 70%, preferably one of 75%, 80%,        85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%        amino acid sequence identity to the amino acid sequence of        NO:221.

In some embodiments the antigen-binding molecule comprises, or consistsof:

-   -   (i) two polypeptides comprising, or consisting of, an amino acid        sequence having at least 70%, preferably one of 75%, 80%, 85%,        90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino        acid sequence identity to the amino acid sequence of NO:222; and    -   (ii) two polypeptides comprising, or consisting of, an amino        acid sequence having at least 70%, preferably one of 75%, 80%,        85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%        amino acid sequence identity to the amino acid sequence of        NO:223.

In some embodiments the antigen-binding molecule comprises, or consistsof:

-   -   (i) two polypeptides comprising, or consisting of, an amino acid        sequence having at least 70%, preferably one of 75%, 80%, 85%,        90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino        acid sequence identity to the amino acid sequence of NO:225, and    -   (ii) two polypeptides comprising, or consisting of, an amino        acid sequence having at least 70%, preferably one of 75%, 80%,        85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%        amino acid sequence identity to the amino acid sequence of        NO:207.

In some embodiments the antigen-binding molecule comprises, or consistsof:

-   -   (i) two polypeptides comprising, or consisting of, an amino acid        sequence having at least 70%, preferably one of 75%, 80%, 85%,        90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino        acid sequence identity to the amino acid sequence of NO:226; and    -   (ii) two polypeptides comprising, or consisting of, an amino        acid sequence having at least 70%, preferably one of 75%, 80%,        85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%        amino acid sequence identity to the amino acid sequence of        NO:207.

In some embodiments the antigen-binding molecule comprises, or consistsof:

-   -   (i) two polypeptides comprising, or consisting of, an amino acid        sequence having at least 70%, preferably one of 75%, 80%, 85%,        90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino        acid sequence identity to the amino acid sequence of NO:227; and    -   (ii) two polypeptides comprising, or consisting of, an amino        acid sequence having at least 70%, preferably one of 75%, 80%,        85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%        amino acid sequence identity to the amino acid sequence of        NO:217.

In some embodiments the antigen-binding molecule comprises, or consistsof:

-   -   (i) two polypeptides comprising, or consisting of, an amino acid        sequence having at least 70%, preferably one of 75%, 80%, 85%,        90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino        acid sequence identity to the amino acid sequence of NO:228; and    -   (ii) two polypeptides comprising, or consisting of, an amino        acid sequence having at least 70%, preferably one of 75%, 80%,        85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%        amino acid sequence identity to the amino acid sequence of        NO:217.

Functional Properties of the Antigen-Binding Molecules

The antigen-binding molecules described herein may be characterised byreference to certain functional properties. In some embodiments, theantigen-binding molecule described herein may possess one or more of thefollowing properties:

-   -   binds to HER3 (e.g. human, mouse, rat or cynomolgus macaque        HER3);    -   does not bind to EGFR and/or HER2;    -   binds to HER3-expressing cells;    -   binds to subdomain II of the extracellular region of HER3;    -   binds to HER3 when HER3 is in open and closed conformations:    -   binds to HER3 independently of NRG;    -   does not compete with MM-121 and/or LJM-716 for binding to HER3;    -   does not compete with M-05-74 and/or M-08-11 for binding to        HER3;    -   inhibits interaction between HER3 and an interaction partner for        HER3 (e.g. HER3, HER2, EGFR, HER4, HGFR, IGF1R and/or cMet);    -   inhibits HER3-mediated signalling;    -   inhibits proliferation of HER3-expressing cells (e.g. in        response to stimulation with NRG);    -   inhibits PI3K/AKT/mTOR and/or MAPK signalling by HER3-expressing        cells (e.g. in response to stimulation with NRG);    -   binds to an activatory Fcγ receptor (e.g. FcγRIIIa);    -   increased binding to an activatory Fcγ receptor;    -   increased binding to an activatory Fcγ receptor as compared to        an equivalent antigen-binding molecule having an Fc region        comprised of CH2-CH3 having the amino acid sequence of SEQ ID        NO:174-175;    -   decreased binding to an inhibitory Fcγ receptor as compared to        an equivalent antigen-binding molecule having an Fc region        comprised of CH2-CH3 having the amino acid sequence of SEQ ID        NO:174-175:    -   increased binding to an activatory Fcγ receptor over an        inhibitory Fcγ receptor as compared to an equivalent        antigen-binding molecule having an Fc region comprised of        CH2-CH3 having the amino acid sequence of SEQ ID NO:174-175;    -   increased or decreased binding to a complement protein (e.g.        C1q) as compared to an equivalent antigen-binding molecule        having an Fc region comprised of CH2-CH3 having the amino acid        sequence of SEQ ID NO:174-175;    -   increased hexamerisation as compared to an equivalent        antigen-binding molecule having an Fc region comprised of        CH2-CH3 having the amino acid sequence of SEQ ID NO:174-175;    -   increased ADCC activity as compared to an equivalent        antigen-binding molecule having an Fc region comprised of        CH2-CH3 having the amino acid sequence of SEQ ID NO:174-175;    -   increased ADCP activity as compared to an equivalent        antigen-binding molecule having an Fc region comprised of        CH2-CH3 having the amino acid sequence of SEQ ID NO:174-175;    -   increased or decreased CDC activity as compared to an equivalent        antigen-binding molecule having an Fc region comprised of        CH2-CH3 having the amino acid sequence of SEQ ID NO:174-175;    -   similar or increased thermostability as compared to an        equivalent antigen-binding molecule having an Fc region        comprised of CH2-CH3 having the amino acid sequence of SEQ ID        NO:174-175;    -   increases killing of HER3-expressing cells;    -   reduces the number/proportion of HER3-expressing cells;    -   and    -   inhibits the development and/or progression of cancer in vivo.

The antigen-binding molecules described herein preferably displayspecific binding to HER3. As used herein, “specific binding” refers tobinding which is selective for the antigen, and which can bediscriminated from non-specific binding to non-target antigen. Anantigen-binding molecule that specifically binds to a target moleculepreferably binds the target with greater affinity, and/or with greaterduration than it binds to other, non-target molecules.

The ability of a given polypeptide to bind specifically to a givenmolecule can be determined by analysis according to methods known in theart, such as by ELISA, Surface Plasmon Resonance (SPR; see e.g. Heartyet al., Methods Mol Biol (2012) 907:411-442). Bio-Layer interferometry(see e.g. Lad et al., (2015) J Biomol Screen 20(4): 498-507), flowcytometry, or by a radiolabeled antigen-binding assay (RIA)enzyme-linked immunosorbent assay. Through such analysis binding to agiven molecule can be measured and quantified. In some embodiments, thebinding may be the response detected in a given assay.

In some embodiments, the extent of binding of the antigen-bindingmolecule to an non-target molecule is less than about 10% of the bindingof the antibody to the target molecule as measured, e.g. by ELISA, SPR,Bio-Layer Interferometry or by RIA. Alternatively, binding specificitymay be reflected in terms of binding affinity where the antigen-bindingmolecule binds with a dissociation constant (K_(D)) that is at least 0.1order of magnitude (i.e. 0.1×10⁰, where n is an integer representing theorder of magnitude) greater than the K_(D) of the antigen-bindingmolecule towards a non-target molecule. This may optionally be one of atleast 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, or 2.0.

In some embodiments, the antigen-binding molecule displays binding tohuman HER3, mouse HER3, rat HER3 and/or cynomolgus macaque (Macacafascicularis) HER3. That is, in some embodiments the antigen-bindingmolecule is cross-reactive for human HER3, mouse HER3, rat HER3 and/orcynomolgus macaque HER3. In some embodiments the antigen-bindingmolecule of the present invention displays cross-reactivity with HER3 ofa non-human primate. Cross-reactivity to HER3 in model species allows invivo exploration of efficacy in syngeneic models without relying onsurrogate molecules.

In some embodiments the antigen-binding molecule binds to human HER3,mouse HER3, rat HER3 and/or cynomolgus macaque HER3; and does not bindto HER2 and/or EGFR (e.g. human HER2 and/or human EGFR).

In some embodiments, the antigen-binding molecule does not displayspecific binding to EGFR human EGFR). In some embodiments, theantigen-binding molecule does not display specific binding to HER2 (e.g.human HER2). In some embodiments, the antigen-binding molecule does notdisplay specific binding to (i.e. does not cross-react with) a member ofthe EGFR family of proteins other than HER3. In some embodiments, theantigen-binding molecule does not display specific binding to EGFR, HER2and/or HER4.

In some embodiments, the antigen-binding molecule of the invention bindsto HER3 (e.g. human HER3) with a K_(D) of 10 μM or less, preferably oneof ≤5 μM, ≤52 μM, ≤1 μM, ≤500 nM, ≤400 nM, ≤300 nM, ≤200 nM, ≤100 nM,≤95 nM, ≤90 nM, ≤85 nM, ≤80 nM, ≤75 nM, ≤70 nM, ≤65 nM, ≤60 nM, ≤55 nM,≤50 nM, ≤45 nM, ≤40 nM, ≤35 nM, ≤30 nM, ≤25 nM, ≤20 nM, ≤15 nM, ≤12.5nM, ≤10 nM, ≤9 nM, ≤3 nM, ≤7 nM, nM, ≤5 nM, ≤4 nM ≤3 nM, ≤2 nM, ≤1 nM,≤900 pM, ≤800 pM, ≤700 pM, ≤600 pM, ≤500 pM, ≤400 pM, ≤300 pM, ≤200 pM,≤100 pM, ≤90 pM, ≤80 pM, ≤70 pM, ≤60 pM, ≤50 pM, ≤40 pM, ≤30 pM, ≤20 pM,≤10 pM, ≤9 pM, ≤8 pM, ≤7 pM, ≤6 pM, ≤5 pM, ≤4 pM, ≤3 pM, ≤2 pM, ≤1 pM.

The antigen-binding molecules of the present invention may bind to aparticular region of interest of HER3. The antigen-binding region of anantigen-binding molecule according to the present invention may bind toa linear epitope of HER3, consisting of a contiguous sequence of aminoacids (i.e. an amino acid primary sequence). In some embodiments, theantigen-binding molecule may bind to a conformational epitope of HER3,consisting of a discontinuous sequence of amino acids of the amino acidsequence.

In some embodiments, the antigen-binding molecule of the presentinvention binds to HER3. In some embodiments, the antigen-bindingmolecule binds to the extracellular region of HER3 (e.g. the regionshown in SEQ ID NO:9). In some embodiments, the antigen-binding moleculebinds to subdomain H of the extracellular region of HER3 (e.g. theregion shown in SEQ ID NO:16).

In some embodiments, the antigen-binding molecule binds to the region ofHER3 shown in SEQ ID NO:229. In some embodiments the antigen-bindingmolecule contacts one or more amino acid residues of the region of HER3shown in SEQ ID NO:229. In some embodiments, the antigen-bindingmolecule binds to the regions of HER3 shown in SEQ ID NOs:230 and 231.In some embodiments the antigen-binding molecule contacts one or moreamino acid residues of the regions of HER3 shown in SEQ ID NOs:230 and231. In some embodiments, the antigen-binding molecule binds to theregion of HER3 shown in SEQ ID NO:230. In some embodiments theantigen-binding molecule contacts one or more amino acid residues of theregion of HER3 shown in SEQ ID NO:230, in some embodiments, theantigen-binding molecule binds to the region of HER3 shown in SEQ IDNO:231. In some embodiments the antigen-binding molecule contacts one ormore amino acid residues of the region of HER3 shown in SEQ ID NO:231.In some embodiments, the antigen-binding molecule binds to the region ofHER3 shown in SEQ ID NO:23. In some embodiments the antigen-bindingmolecule contacts one or more amino acid residues of the region of HER3shown in SEQ ID NO:23. In some embodiments, the antigen-binding moleculebinds to the region of HER3 shown in SEQ ID NO:21. In some embodimentsthe antigen-binding molecule contacts one or more amino acid residues ofthe region of HER3 shown in SEQ ID NO:21. In some embodiments theantigen-binding molecule binds to the region of HER3 shown in SEQ IDNO:19. In some embodiments the antigen-binding molecule contacts one ormore amino acid residues of the region of HER3 shown in SEQ ID NO:19. Insome embodiments, the antigen-binding molecule binds to the region ofHER3 shown in SEQ ID NO:22. In some embodiments the antigen-bindingmolecule contacts one or more amino acid residues of the region of HER3shown in SEQ ID NO:22.

In some embodiments, the antigen-binding molecule of the presentinvention is capable of binding to a polypeptide comprising, orconsisting of, the amino acid sequence of one of SEQ ID NOs:1, 3, 4, 6or 8. In some embodiments, the antigen-binding molecule is capable ofbinding to a polypeptide comprising, or consisting of, the amino acidsequence of SEQ ID NO:9. In some embodiments, the antigen-bindingmolecule is capable of binding to a polypeptide comprising, orconsisting of, the amino acid sequence of SEQ ID NO:16. In someembodiments, the antigen-binding molecule is capable of binding to apeptide/polypeptide comprising, or consisting of, the amino acidsequence of SEQ ID NO:229. In some embodiments, the antigen-bindingmolecule is capable of binding to a peptide/polypeptide comprising, orconsisting of, the amino acid sequences of SEQ ID NO230 and 231. In someembodiments, the antigen-binding molecule is capable of binding to apeptide/polypeptide comprising, or consisting of, the amino acidsequence of SEQ ID NO:230. In some embodiments, the antigen-bindingmolecule is capable of binding to a peptide/polypeptide comprising, orconsisting of, the amino acid sequence of SEQ ID NO:231. In someembodiments, the antigen-binding molecule is capable of binding to apeptide/polypeptide comprising, or consisting of, the amino acidsequence of SEQ ID NO:23. In some embodiments, the antigen-bindingmolecule is capable of binding to a peptide/polypeptide comprising, orconsisting of, the amino acid sequence of SEQ ID NO:21. In someembodiments, the antigen-binding molecule is capable of binding to apeptide/polypeptide comprising, or consisting of, the amino acidsequence of SEQ ID NO:19. In some embodiments, the antigen-bindingmolecule is capable of binding to a peptide/polypeptide comprising, orconsisting of, the amino acid sequence of SEQ ID NO:22.

In some embodiments, the antigen-binding molecule does not bind to theregion of HER3 corresponding to positions 260 to 279 of SEQ ID NO:1. Insome embodiments the antigen-binding molecule does not contact an aminoacid residue of the region of HER3 corresponding to positions 260 to 279of SEQ ID NO:1. In some embodiments, the antigen-binding molecule doesnot bind to the region of HER3 shown in SEQ ID NO:23. In someembodiments the antigen-binding molecule does not contact an amino acidresidue of the region of HER3 shown in SEQ ID NO:23. In someembodiments, the antigen-binding molecule is not capable of binding to apeptide consisting of the amino acid sequence corresponding to positions260 to 279 of SEQ ID NO:1. In some embodiments, the antigen-bindingmolecule is not capable of binding to a peptide consisting of the aminoacid sequence of SEQ ID NO:23.

As used herein, a “peptide” refers to a chain of two or more amino acidmonomers linked by peptide bonds. A peptide typically has a length inthe region of about 2 to 50 amino acids. A “polypeptide” is a polymerchain of two or more peptides. Polypeptides typically have a lengthgreater than about 50 amino acids.

The ability of an antigen-binding molecule to bind to a givenpeptide/polypeptide can be analysed by methods well known to the skilledperson, including analysis by ELISA, immunoblot (e.g. western blot),immunoprecipitation, surface plasmon resonance and biolayerinterferometry.

Ligand binding to HER3 promotes conformational changes that enables HER3to homo- or heterodimerise, resulting in activation of downstreampathways. HER3 demonstrates ‘closed’ and ‘open’ conformations. By closedconformation it is meant that HER3 is in a tethered conformation and isunavailable for receptor homo- or heterodimerisation. By openconformation it is meant that HER3 is in an extended conformation and isavailable for receptor homo- or heterodimerisation.

In some embodiments the antigen-binding molecule is capable of bindingto HER3 when HER3 is in the open conformation. In some embodiments theantigen-binding molecule is capable of binding to HER3 when HER3 is inthe closed conformation. In some embodiments the antigen-bindingmolecule is capable of binding to HER3 when HER3 is in the open and/orclosed conformation. In some embodiments the antigen-binding molecule iscapable of binding to the HER3 ectodomain when HER3 is in the openand/or closed conformation. In some embodiments the antigen-bindingmolecule is capable of binding to the HER3 dimerisation arm when HER3 isin the open and/or closed conformation. Binding to the dimerisation armenables an antigen-binding molecule to prevent interaction between HER3and an interaction partner for HER3, e.g. as described herein.

In some embodiments the antigen-binding molecule is capable of bindingto HER3 in the presence and/or absence of a ligand for HER3. In someembodiments the antigen-binding molecule is capable of binding to HER3independently of a ligand for HER3. In some embodiments the ligand isNRG, NRG-1 and/or NRG-2 HER3 is activated by ligand binding to itsextracellular domain which promotes conformational changes that enablesHER3 to homo- or heterodimerise. Binding of an antigen-binding moleculeto HER3 independently of ligand binding allows the antigen-bindingmolecule to inhibit the action of HER3 in both ligand-absent andligand-present conformational states. In some embodiments theantigen-binding molecule does not compete with ligand binding to HER3.In some embodiments the antigen-binding molecule does not bind to HER3at the ligand binding site.

In some embodiments the antigen-binding molecule is capable of bindingthe same region of HER3, or an overlapping region of HER3, to the regionof HER3 which is bound by an antibody comprising the VH and VL sequencesof one of clones 10D1, 10D1_c75, 10D1_c76, 10D1_c77, 10D1_c78v1,10D1_c78v2, 10D1_11E, 10D1_c85v1, 10D1_c85v2, 10D1_c85o1, 10D1_c85o2,10D1_c87, 10D1_c89, 10D1_c90, 10D1_c91, 10D1_c92, 10D1_c93, 10A6,4-35-B2 or 4-35-B4. In some embodiments the antigen-binding molecule iscapable of binding the same region of HER3, or an overlapping region ofHER3, to the region of HER3 which is bound by an antibody comprising theVH and VL sequences of one of clones 10D1_c89, 10D1_c90 or 10D1_c91. Insome embodiments the antigen-binding molecule is capable of binding thesame region of HER3, or an overlapping region of HER3, to the region ofHER3 which is bound by an antibody comprising the VH and VL sequences ofclone 10D1_c59.

The region of a peptide/polypeptide to which an antibody binds can bedetermined by the skilled person using various methods well known in theart, including X-ray co-crystallography analysis of antibody-antigencomplexes, peptide scanning, mutagenesis mapping, hydrogen-deuteriumexchange analysis by mass spectrometry, phage display, competition ELISAand proteolysis-based ‘protection’ methods. Such methods are described,for example, in Gershoni et al., BioDrugs, 2007, 21(3):145-156, which ishereby incorporated by reference in its entirety. Such methods can alsobe used to determine whether an antigen-binding molecule is capable ofbinding to proteins in different conformations.

In some embodiments the antigen-binding molecule of the presentinvention does not bind to HER3 in the same region of HER3, or anoverlapping region of HER3, as an antibody comprising the VH and VLsequences of anti-HER3 antibody clone MM-121 (described e.g. inSchoeberl et al., Sci. Signal. (2009) 2(77): ra31) and/or LJM-716(described e.g. Garner et al., Cancer Res (2013) 73: 6024-6035). In someembodiments the antigen-binding molecule of the present invention doesnot display competition with an antibody comprising the VH and VLsequences of anti-HER3 antibody clone MM-121 and/or LJM-716 for bindingto HER3, e.g. as determined by SPR analysis.

In some embodiments the antigen-binding molecule of the presentinvention binds to HER3 in a region which is accessible to anantigen-binding molecule (i.e., an extracellular antigen-bindingmolecule) when HER3 is expressed at the cell surface (i.e. in or at thecell membrane). In some embodiments the antigen-binding molecule iscapable of binding to HER3 expressed at the cell surface of a cellexpressing HER3. In some embodiments the antigen-binding molecule iscapable of binding to HER3-expressing cells (e.g. HER3+ cells, e.g.HER3+ cancer cells).

The ability of an antigen-binding molecule to bind to a given cell typecan be analysed by contacting cells with the antigen-binding molecule,and detecting antigen-binding molecule bound to the cells, e.g. after awashing step to remove unbound antigen-binding molecule. The ability ofan antigen-binding molecule to bind to immune cell surfacemolecule-expressing cells and/or cancer cell antigen-expressing cellscan be analysed by methods such as flow cytometry and immunofluorescencemicroscopy.

The antigen-binding molecule of the present invention may be anantagonist of HER3. In some embodiments, the antigen-binding molecule iscapable of inhibiting a function or process (e.g. interaction,signalling or other activity) mediated by HER3 and/or a binding partnerfor HER3 (e.g. HER3 (i.e. in the case of homodimerisation), HER2, EGFR,HER4, HGFR, IGF1R and/or cMet). Herein, ‘inhibition’ refers to areduction, decrease or lessening relative to a control condition.

In some embodiments the antigen-binding molecule of the presentinvention is capable of inhibiting interaction between HER3 and aninteraction partner for HER3. An interaction partner for HER3 may beexpressed by the same cell as the HER3. An interaction partner or HER3may be expressed at the cell surface (i.e. in or at the cell membrane).In some embodiments an interaction partner for HER3 may be a member ofthe EGFR family of proteins, e.g. HER3, HER2, EGFR, HER4, HGFR, IGF1Rand/or cMet. In some embodiments an interaction partner for HER3 may beIGF1R and/or cMet. Interaction between HER3 and an interaction partnerfor HER3 may result in the formation of a polypeptide complex.Interaction between HER3 and an interaction partner for HER3 to form apolypeptide complex may be referred to as multimerisation. Wheremultimerisation is between polypeptide monomers multimerisation may bereferred to as dimerisation.

In some embodiments the antigen-binding molecule is capable ofinhibiting interaction between HER3 monomers. In some embodiments theantigen-binding molecule is capable of inhibiting interaction betweenHER3 and HER2. In some embodiments the antigen-binding molecule iscapable of inhibiting interaction between HER3 and EGFR. In someembodiments the antigen-binding molecule is capable of inhibitinginteraction between HER3 and HER4. In some embodiments theantigen-binding molecule is capable of inhibiting interaction betweenHER3 and HGFR. In some embodiments the antigen-binding molecule iscapable of inhibiting interaction between HER3 and IGF1R. In someembodiments the antigen-binding molecule is capable of inhibitinginteraction between HER3 and cMet.

Inhibition of interaction may be achieved by binding of theantigen-binding molecule to a region of HER3 required for interactionbetween HER3 and an interaction partner for HER3 (e.g. the dimerisationloop of HER3 shown in SEQ ID NO:19). In some embodiments theantigen-binding molecule contacts one or more residues of HER3 necessaryfor interaction between HER3 and an interaction partner for HER3; inthis way the antigen-binding molecule makes the region unavailable,thereby inhibiting interaction. In some embodiments the antigen-bindingmolecule binds to HER3 in a manner which inhibits/prevents interactionbetween HER3 and an interaction partner for HER3. In some embodimentsthe antigen-binding molecule inhibits/prevents access of the interactionpartner for HER3 to the region of HER3 required for interaction betweenHER3 and the interaction partner for HER3; this may be achieved in caseseven where the antigen-binding molecule does not contact the region ofHER3 required for interaction between HER3 and the interaction partnerfor HER3, e.g. through steric inhibition of access of the interactionpartner for HER3 to the region of HER3 required for interaction betweenHER3 and the interaction partner.

In some embodiments the antigen-binding molecule is capable ofinhibiting homodimerisation of HER3 monomers. In some embodiments theantigen-binding molecule is capable of inhibiting dimerisation betweenHER3 and HER2. In some embodiments the antigen-binding molecule iscapable of inhibiting dimerisation between HER3 and EGFR. In someembodiments the antigen-binding molecule is capable of inhibitingdimerisation between HER3 and HER4. In some embodiments theantigen-binding molecule is capable of inhibiting dimerisation betweenHER3 and HGFR. In some embodiments the antigen-binding molecule iscapable of inhibiting dimerisation between HER3 and IGF1R. In someembodiments the antigen-binding molecule is capable of inhibitingdimerisation between HER3 and cMet.

The ability of an antigen-binding molecule to inhibit interactionbetween two factors can be determined for example by analysis ofinteraction in the presence of, or following incubation of one or bothof the interaction partners with, the antibody/fragment. Assays fordetermining whether a given antigen-binding molecule is capable ofinhibiting interaction between two interaction partners includecompetition ELISA assays and analysis by SPR. In some embodiments theantigen-binding molecule is a competitive inhibitor of interactionbetween HER3 and an interaction partner for HER3.

In some embodiments, the antigen-binding molecule of the presentinvention is capable of inhibiting interaction between HER3 and aninteraction partner for HER3 (e.g. HER3, HER2, EGFR, HER4, HGFR, IGF1Rand/or cMet) to less than less than 1 times, e.g. ≤0.99 times, ≤0.95times, ≤0.9 times, ≤0.85 times, ≤0.8 times, ≤0.75 times, ≤0.7 times,≤0.65 times, ≤0.6 times, ≤0.55 times, ≤0.5 times, ≤0.45 times, ≤0.4times, ≤0.35 times, ≤0.3 times, ≤0.25 times, ≤0.2 times, ≤0.15 times,≤0.1 times, ≤0.05 times, or ≤0.01 times the level of interaction betweenHER3 and the interaction partner for HER3 in the absence of theantigen-binding molecule (or in the presence of an appropriate controlantigen-binding molecule), in a suitable assay.

The ability of an antigen-binding molecule to inhibit interactionbetween interaction partners can also be determined by analysis of thedownstream functional consequences of such interaction. For example,downstream functional consequences of interaction between HER3 andinteraction partners for HER3 include PI3K/AKT/mTOR and/or MAPKsignalling. For example, the ability of an antigen-binding molecule toinhibit interaction of HER3 and an interaction partner for HER3 may bedetermined by analysis of PI3K/AKT/mTOR and/or MAPK signalling followingtreatment with NRG in the presence of the antigen-binding molecule.PI3K/AKT/mTOR and/or MAPK signalling can be detected and quantified e.g.using antibodies capable of detecting phosphorylated members of thesignal transduction pathways.

The ability of an antigen-binding molecule to inhibit interaction ofHER3 and an interaction partner for HER3 can also be determined byanalysing proliferation of cells expressing HER3 following treatmentwith NRG in the presence of the antigen-binding molecule. Cellproliferation can be determined e.g. by detecting changes in number ofcells over time, or by in vitro analysis of incorporation of³H-thymidine or by CFSE dilution assay, e.g. as described in Fulcher andWong, Immunol Cell Biol (1999) 77(6): 559-564, hereby incorporated byreference in entirety.

In some embodiments, the antigen-binding molecule of the presentinvention is capable of inhibiting proliferation of cells harbouringmutation to BRAF V600, e.g. cells comprising the BRAF V600E or V600Kmutation (see Example 10).

In some embodiments the antigen-binding molecule inhibits HER3-mediatedsignalling. HER3-mediated signalling can be analysed e.g. using an assayof a correlate of HER3-mediated signalling, e.g. cell proliferation,and/or phosphorylation of one or more signal transduction molecules ofthe PI3K/AKT/mTOR and/or MAPK signal transduction pathways.

In some embodiments, the antigen-binding molecule of the presentinvention is capable of inhibiting PI3K/AKT/mTOR and/or MAPK signallingby HER3-expressing cells. The level of PI3K/AKT/mTOR and/or MAPKsignalling may be analysed by detection and quantification of the levelof phosphorylation of one or more of the components of the PI3K/AKT/mTORand/or MAPK pathways, e.g. following stimulation with NRG (see Example4.3).

In some embodiments, the antigen-binding molecule of the presentinvention is capable of inhibiting proliferation of HER3-expressingcells, e.g. in response to stimulation with NRG. In some embodiments,the antigen-binding molecule of the present invention is capable ofinhibiting proliferation of HER3-expressing cells to less than less than1 times, e.g. ≤0.99 times, ≤0.95 times, ≤0.9 times, ≤0.85 times, ≤0.8times, ≤0.75 times, ≤0.7 times, ≤0.65 times, ≤0.6 times, ≤0.55 times,≤0.5 times, ≤0.45 times, ≤0.4 times, ≤0.35 times, ≤0.3 times, ≤0.25times, ≤0.2 times, ≤0.15 times, ≤0.1 times, ≤0.05 times, or ≤0.01 timesthe level of proliferation of HER3-expressing cells in the absence ofthe antigen-binding molecule (or in the presence of an appropriatecontrol antigen-binding molecule), in a suitable assay.

In some embodiments, the antigen-binding molecule of the presentinvention is capable of inhibiting PI3K/AKT/mTOR and/or MAPK signallingby HER3-expressing cells to less than less than 1 times, e.g. ≤0.99times, ≤0.95 times, ≤0.9 times, ≤0.85 times, ≤0.8 times, ≤0.75 times,≤0.7 times, ≤0.65 times, ≤0.6 times, ≤0.55 times, ≤0.5 times, ≤0.45times, ≤0.4 times, ≤0.35 times, ≤0.3 times, ≤0.25 times, ≤0.2 times,≤0.15 times, ≤0.1 times, ≤0.05 times, or ≤0.01 times the level ofsignalling by HER3-expressing cells in the absence of theantigen-binding molecule (or in the presence of an appropriate controlantigen-binding molecule), in a suitable assay.

HER3-mediated signalling can be investigated in vitro, e.g. as describedin Example 8.9, or in vivo, e.g. as described in Example 11.

ADCC activity can be analysed e.g. according to the methods described inYamashita et al., Scientific Reports (2016) 6:19772 (hereby incorporatedby reference in its entirety), or by ⁵¹Cr release assay as describede.g. in Jedema et al., Blood (2004) 103: 2677-82 (hereby incorporated byreference in its entirety). ADCC activity can also be analysed using thePierce LDH Cytotoxicity Assay Kit, in accordance with the manufacturer'sinstructions (as described in Example 5 herein).

ADCP can be analysed e.g. according to the method described in Kamen etal., J Immunol (2017) 198 (1 Supplement) 157.17 (hereby incorporated byreference in its entirety).

The ability to induce CDC can be analysed e.g. using a C1q bindingassay, e.g. as described in Schlothauer et al., Protein Engineering,Design and Selection (2016), 29(10):457-466 (hereby incorporated byreference in its entirety).

Thermostability of antigen-binding molecules can be analysed by methodswell known to the skilled person, including Differential ScanningFluorimetry and Differential Scanning calorimetry (DSC), which aredescribed e.g. in He et al., J Pharm Sci. (2010) which is herebyincorporated by reference in its entirety. Thermostability may bereflected in terms of a melting temperature (T_(m)), unfoldingtemperature or disassembly temperature (expressed e.g. in ° C. or ° F.).

In some embodiments, an antigen-binding molecule comprising an Fc regionas described herein binds to an activatory Fcγ receptor (e.g. hFcγRIIa(e.g. hFcγRIIa167H, hFcγRIIa167R), hFcγRIIIa (e.g. hFcγRIIIa158V,hFcγRIIIa158F), mFcγRIV, mFcγRIII) with an affinity of binding which isgreater than 1 times, e.g. greater than 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,or greater than 20 times the affinity of binding to the activatory Fcγreceptor by an equivalent antigen-binding molecule having an Fc regioncomprised of CH2-CH3 having the amino acid sequence of SEQ IDNO:174-175. In some embodiments the K_(D), of the antigen-bindingmolecule comprising an Fc region described herein for binding to theactivatory Fcγ receptor is less than 1 times, e.g. less than 0.9, 0.8,0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06 or less than0.05 times the K_(D) of an equivalent antigen-binding molecule having anFc region comprised of CH2-CH3 having the amino acid sequence of SEQ IDNO:174-175 for the activatory Fcγ receptor.

In some embodiments, the antigen-binding molecule comprising an Fcregion as described herein binds to an activatory Fcγ receptor (e.g.hFcγRIIa (e.g. hFcγRIIa167H, hFcγRIIa167R), hFcγRIIIa (e.g.hFcγRIIIa158V, hFcγRIIIa158F), mFcγRIV, mFcγRIII) with a K_(D) of 1000nM or less, preferably one of ≤500 nM, ≤100 nM, ≤75 nM, ≤50 nM, ≤40 nM,≤30 nM, ≤20 nM, ≤15 nM, ≤12.5 nM, ≤10 nM, ≤9 nM, nM, ≤7 nM, ≤6 nM, ≤5nM, ≤4 nM ≤3 nM, ≤2 nM or ≤1 nM.

In some embodiments, an antigen-binding molecule comprising an Fc regionas described herein binds to an FcRn (e.g. hFcRn, mFcRn) with anaffinity of binding which is greater than 1 times, e.g. greater than 2,3, 4, 5, 6, 7, 8, 9, 10, 15, or greater than 20 times the affinity ofbinding to the FcRn by an equivalent antigen-binding molecule having anFc region comprised of CH2-CH3 having the amino acid sequence of SEQ IDNO:174-175. In some embodiments the K_(D) of the antigen-bindingmolecule comprising an Fc region described herein for binding to theFcRn is less than 1 times, e.g. less than 0.9, 0.8, 0.7, 0.6, 0.5, 0.4,0.3, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06 or less than 0.05 times the K_(D)of an equivalent antigen-binding molecule having an Fc region comprisedof CH2-CH3 having the amino acid sequence of SEQ ID NO:174-175 for theFcRn.

In some embodiments, the antigen-binding molecule comprising an Fcregion as described herein binds to an FcRn (e.g. hFcRn, mFcRn) with aK_(D) of 1000 nM or less, preferably one of ≤500 nM, ≤100 nM, ≤75 nM,≤50 nM, ≤40 nM, ≤30 nM, ≤20 nM, ≤15 nM, ≤12.5 nM, ≤10 nM, ≤9 nM, ≤8 nM,≤7 nM, ≤6 nM, ≤5 nM, ≤4 nM ≤3 nM, ≤2 nM or ≤1 nM.

In some embodiments, an antigen-binding molecule comprising an Fc regionas described herein binds to an inhibitory Fcγ receptor (e.g. hFcγRIIbmFcγRIIb) with an affinity of binding which is less than 1 times, e.g.less than 0.9, 0.8, 0.7, 0.8, 0.5, 0.4, 0.3, 0.2, or less than 0.1 timesthe affinity of binding to the inhibitory Fcγ receptor by an equivalentantigen-binding molecule having an Fc region comprised of CH2-CH3 havingthe amino acid sequence of SEQ ID NO:174-175. In some embodiments theK_(D) of the antigen-binding molecule comprising an Fc region describedherein for binding to the inhibitory Fcγ receptor is greater than 1times, e.g. greater than 2, 3, 4, 5, 6, 7, 8, 9 or greater than 10 timesthe K_(D) of an equivalent antigen-binding molecule having an Fc regioncomprised of CH2-CH3 having the amino acid sequence of SEQ ID NO:174-175for the inhibitory Fcγ receptor.

In some embodiments, the antigen-binding molecule comprising an Fcregion as described herein binds to an inhibitory Fcγ receptor (e.g.hFcγRIIb mFcγRIIb) with a K_(D) 1 nM or greater, preferably one of ≥5nM, ≥10 nM, ≥50 nM, ≥100 nM, ≥500 nM, ≥1000 nM, ≥2000 nM, ≥3000 nM,≥4000 nM or ≥5000 nM.

In some embodiments the selectivity of binding for an activatory Fcγreceptor (e.g. hFcγRIIa; relative to an inhibitory Fcγ receptor (e.g.hFcγRIIb) for an antigen-binding molecule comprising an Fc region asdescribed herein is greater than 1 times, e.g. greater than 2, 3, 4, 5,6, 7, 8, 9, 10, 15, or greater than 20 times selectivity of bindingdisplayed by an equivalent antigen-binding molecule having an Fc regioncomprised of CH2-CH3 having the amino acid sequence of SEQ IDNO:174-175.

In some embodiments, an antigen-binding molecule comprising an Fc regionas described herein displays ADCC which is greater than 1 times, e.g.greater than 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or greater than 20 timesthe ADCC displayed by an equivalent antigen-binding molecule having anFc region comprised of CH2-CH3 having the amino acid sequence of SEQ IDNO:174-175.

In some embodiments, the EC50 (ng/mil determined for an antigen-bindingmolecule comprising an Fc region as described herein in an assay of ADCCactivity less than 1 times, e.g. less than 0.9, 0.8, 0.7, 0.6, 0.5, 0.4,0.3, 0.2, or less than 0.1 times the EC50 determined for an equivalentantigen-binding molecule having an Fc region comprised of CH2-CH3 havingthe amino acid sequence of SEQ ID NO:174-175.

In some embodiments, the EC50 (ng/ml) for an antigen-binding moleculecomprising an Fc region as described herein in an assay of ADCC activityis 500 ng/ml or less, preferably one of ≤400 ng/ml, ≤300 ng/ml, ≤200ng/ml, ≤100 ng/ml, ≤90 ng/ml, ≤80 ng/ml, ≤70 ng/ml, ≤60 ng/ml, ≤50ng/ml, ≤40 ng/ml, ≤30 ng/ml, ≤20 ng/ml, or ≤10 ng/ml.

In some embodiments, an antigen-binding molecule comprising an Fc regionas described herein may have a melting temperature, unfoldingtemperature or disassembly temperature which is which is ≥0.75 times and≤1.25 times, e.g. ≥0.8 times and ≤1.2 times, ≥0.85 times and ≤1.15times, ≥0.9 times and ≤1.1 times, ≥0.91 times and ≤1.09 times, ≥0.92times and ≤1.08 times, ≥0.93 times and ≤1.07 times, ≥0.94 times and≤1.06 times, ≥0.95 times and ≤1.05 times, ≥0.96 times and ≤1.04 times,≥0.97 times and ≤1.03 times, ≥0.98 times and ≤1.02 times, or ≥0.99 timesand ≤1.01 times the melting temperature, unfolding temperature ordisassembly temperature of an equivalent antigen-binding molecule havingan Fc region comprised of CH2-CH3 having the amino acid sequence of SEQID NO:174-175.

In some embodiments, the antigen-binding molecule of the presentinvention is capable of increasing killing of HER3-expressing cells.Killing of HER3-expressing cells may be increased through an effectorfunction of the antigen-binding molecule. In embodiments whereinantigen-binding molecule comprises an Fc region the antigen-bindingmolecule may increasing killing of HER3-expressing cells through one ormore of complement dependent cytotoxicity (CDC), antibody-dependentcell-mediated cytotoxicity (ADCC) and antibody-dependent cellularphagocytosis (ADCP).

An antigen-binding molecule which is capable of increasing killing ofHER3-expressing cells can be identified by observation of an increasedlevel of killing of HER3-expressing cells in the presence of orfollowing incubation of the HER3-expressing cells with theantigen-binding molecule, as compared to the level of cell killingdetected in the absence of the antigen-binding molecule (or in thepresence of an appropriate control antigen-binding molecule), in anappropriate assay. Assays of CDC, ADCC and ADCP are well known theskilled person. The level of killing of HER3-expressing cells can alsobe determined by measuring the number/proportion of viable and/ornon-viable HER3-expressing cells following exposure to differenttreatment conditions.

In some embodiments, the antigen-binding molecule of the presentinvention is capable of increasing killing of HER3-expressing cells(e.g. HER3-expressing cancer cells) to more than 1 times, e.g. ≥1.01times, ≥1.02 times, ≥1.03 times, ≥1.04 times, ≥1.05 times, ≥1.1 times,≥1.2 times, ≥1.3 times, ≥1.4 times, ≥1.5 times, ≥1.6 times, ≥1.7 times,≥1.8 times, ≥1.9 times, ≥2 times, ≥3 times, ≥4 times, ≥5 times, ≥6times, ≥7 times, ≥8 times, ≥9 times or ≥10 times the level of killingobserved in the absence of the antigen-binding molecule (or in thepresence of an appropriate control antigen-binding molecule).

In some embodiments, the antigen-binding molecule of the presentinvention is capable of reducing the number of HER3-expressing cells(e.g. HER3-expressing cancer cells) to less than less than 1 times, e.g.≤0.99 times, ≤0.95 times, ≤0.9 times, ≤0.85 times, ≤0.8 times, ≤0.75times, ≤0.7 times, ≤0.65 times, ≤0.6 times, ≤0.55 times, ≤0.5 times,≤0.45 times, ≤0.4 times, ≤0.35 times, ≤0.3 times, ≤0.25 times, ≤0.2times, ≤0.15 times, ≤0.1 times, ≤0.05 times, or ≤0.01 times the numberof HER3-expressing cells (e.g. HER3-expressing cancer cells) detectedfollowing incubation in the absence of the antigen-binding molecule (orfollowing incubation in the presence of an appropriate controlantigen-binding molecule), in a comparable assay.

In some embodiments, the antigen-binding molecule of the presentinvention inhibits the development and/or progression of cancer in vivo.

In some embodiments the antigen-binding molecule causes an increase inthe killing of cancer cells, by effector immune cells. In someembodiments the antigen-binding molecule causes a reduction in thenumber of cancer cells in vivo, e.g. as compared to an appropriatecontrol condition. In some embodiments the antigen-binding moleculeinhibits tumor growth, e.g. as determined by measuring tumor size/volumeover time.

The antigen-binding molecule of the present invention may be analysedfor the ability to inhibit development and/or progression of cancer inan appropriate in vivo model, e.g. cell line-derived xenograft model.The cell line-derived xenograft model may be derived fromHER3-expressing cancer cells. In some embodiments the model is an N87cell-derived model, a SNU16 cell-derived model, a FaDu cell-derivedmodel, an OvCAR8 cell-derived model, a HCC95 cell-derived model, an A549cell-derived model, an ACHN cell-derived model or a HT29 cell-derivedmodel.

The cancer may be a HER3-associated cancer as described herein (i.e.cancers for which HER3 gene/protein expression is a risk factor for,and/or is positively associated with, the onset, development,progression or severity of symptoms of the cancer, and/or metastasis).The cancer may comprise HER3-expressing cells. In some embodiments thecancer comprises a HER3+ tumor.

In some embodiments, administration of an antigen-binding moleculeaccording to the present invention may cause one or more of: inhibitionof the development/progression of the cancer, a delay to/prevention ofonset of the cancer, a reduction in/delay to/prevention of tumor growth,a reduction in/delay to/prevention of metastasis, a reduction in theseverity of the symptoms of the cancer, a reduction in the number ofcancer cells, a reduction in tumour size/volume, and/or an increase insurvival (e.g. progression free survival), e.g. as determined in anappropriate HER3-expressing cancer cell line-derived xenograft model.

In some embodiments, the antigen-binding molecule of the presentinvention is capable of inhibiting tumor growth in a HER3-expressingcancer cell line-derived xenograft model to less than less than 1 times,e.g. ≤0.99 times, ≤0.95 times, ≤0.9 times, ≤0.85 times, ≤0.8 times,≤0.75 times, ≤0.7 times, ≤0.65 times, ≤0.6 times, ≤0.55 times, ≤0.5times, ≤0.45 times, ≤0.4 times, ≤0.35 times, ≤0.3 times, ≤0.25 times,≤0.2 times, ≤0.15 times, ≤0.1 times, ≤0.05 times, or ≤0.01 times thetumor growth observed in the absence of treatment with theantigen-binding molecule (or following treatment with an appropriatenegative control antigen-binding molecule).

Chimeric Antigen Receptors (CARs)

The present invention also provides Chimeric Antigen Receptors (CARs)comprising the antigen-binding molecules or polypeptides of the presentinvention.

CARs are recombinant receptors that provide both antigen-binding and Tcell activating functions. CAR structure and engineering is reviewed,for example, in Dotti et al., Immunol Rev (2014) 257(1), herebyincorporated by reference in its entirety. CARs comprise anantigen-binding region linked to a cell membrane anchor region and asignalling region. An optional hinge region may provide separationbetween the antigen-binding region and cell membrane anchor region, andmay act as a flexible linker.

The CAR of the present invention comprises an antigen-binding regionwhich comprises or consists of the antigen-binding molecule of thepresent invention, or which comprises or consists of a polypeptideaccording to the invention.

The cell membrane anchor region is provided between the antigen-bindingregion and the signalling region of the CAR and provides for anchoringthe CAR to the cell membrane of a cell expressing a CAR, with theantigen-binding region in the extracellular space, and signalling regioninside the cell. In some embodiments, the CAR comprises a cell membraneanchor region comprising or consisting of an amino acid sequence whichcomprises, consists of, or is derived from, the transmembrane regionamino acid sequence for one of CD3-ζ, CD4, CD8 or CD28. As used herein,a region which is ‘derived from’ a reference amino acid sequencecomprises an amino acid sequence having at least 60%, e.g. one of atleast 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or 100% sequence identity to the reference sequence.

The signalling region of a CAR allows for activation of the T cell. TheCAR signalling regions may comprise the amino acid sequence of theintracellular domain of CD3-ζ, which provides immunoreceptortyrosine-based activation motifs (ITAMs) for phosphorylation andactivation of the CAR-expressing T cell. Signalling regions comprisingsequences of other ITAM-containing proteins such as FcγRI have also beenemployed in CARs (Haynes et al., 2001 J Immunol 166(1):182-187).Signalling regions of CARs may also comprise co-stimulatory sequencesderived from the signalling region of co-stimulatory molecules, tofacilitate activation of CAR-expressing T cells upon binding to thetarget protein. Suitable co-stimulatory molecules include CD28, OX40,4-1BB, ICOS and CD27. In some cases CARs are engineered to provide forco-stimulation of different intracellular signalling pathways. Forexample, signalling associated with CD28 costimulation preferentiallyactivates the phosphatidylinositol 3-kinase (P13K) pathway, whereas the4-1BB-mediated signalling is through TNF receptor associated factor(TRAF) adaptor proteins. Signalling regions of CARs therefore sometimescontain co-stimulatory sequences derived from signalling regions of morethan one co-stimulatory molecule. In some embodiments, the CAR of thepresent invention comprises one or more co-stimulatory sequencescomprising or consisting of an amino acid sequence which comprises,consists of, or is derived from, the amino acid sequence of theintracellular domain of one or more of CD28, OX40, 4-1BB, ICOS and CD27.

An optional hinge region may provide separation between theantigen-binding domain and the transmembrane domain, and may act as aflexible linker. Hinge regions may be derived from IgG1. In someembodiments, the CAR of the present invention comprises a hinge regioncomprising or consisting of an amino acid sequence which comprises,consists of, or is derived from, the amino acid sequence of the hingeregion of IgG1.

Also provided is a cell comprising a CAR according to the invention. TheCAR according to the present invention may be used to generateCAR-expressing immune cells, e.g. CAR-T or CAR-NK cells. Engineering ofCARS into immune cells may be performed during culture, in vitro.

The antigen-binding region of the CAR of the present invention may beprovided with any suitable format, e.g. scFv, scFab, etc.

Nucleic Acids and Vectors

The present invention provides a nucleic acid, or a plurality of nucleicacids, encoding an antigen-binding molecule, polypeptide or CARaccording to the present invention.

In some embodiments, the nucleic acid is purified or isolated, e.g. fromother nucleic acid, or naturally-occurring biological material. In someembodiments the nucleic acid(s) comprise or consist of DNA and/or RNA.

The present invention also provides a vector, or plurality of vectors,comprising the nucleic acid or plurality of nucleic acids according tothe present invention.

The nucleotide sequence may be contained in a vector, e.g. an expressionvector. A “vector” as used herein is a nucleic acid molecule used as avehicle to transfer exogenous nucleic acid into a cell. The vector maybe a vector for expression of the nucleic acid in the cell. Such vectorsmay include a promoter sequence operably linked to the nucleotidesequence encoding the sequence to be expressed. A vector may alsoinclude a termination codon and expression enhancers. Any suitablevectors, promoters, enhancers and termination codons known in the artmay be used to express a peptide or polypeptide from a vector accordingto the invention.

The term “operably linked” may include the situation where a selectednucleic acid sequence and regulatory nucleic acid sequence (e.g.promoter and/or enhancer) are covalently linked in such a way as toplace the expression of nucleic acid sequence under the influence orcontrol of the regulatory sequence (thereby forming an expressioncassette). Thus a regulatory sequence is operably linked to the selectednucleic acid sequence if the regulatory sequence is capable of effectingtranscription of the nucleic acid sequence. The resulting transcripts)may then be translated into a desired peptide(s)/polypeptide(s).

Suitable vectors include plasmids, binary vectors, DNA vectors, mRNAvectors, viral vectors (e.g. gammaretroviral vectors (e.g. murineLeukemia virus (MLV)-derived vectors), lentiviral vectors, adenovirusvectors, adeno-associated virus vectors, vaccinia virus vectors andherpesvirus vectors), transposon-based vectors, and artificialchromosomes (e.g. yeast artificial chromosomes).

In some embodiments, the vector may be a eukaryotic vector, e.g. avector comprising the elements necessary for expression of protein fromthe vector in a eukaryotic cell. In some embodiments, the vector may bea mammalian vector, e.g. comprising a cytomegalovirus (CMV) or SV40promoter to drive protein expression.

Constituent polypeptides of an antigen-binding molecule according to thepresent invention may be encoded by different nucleic acids of theplurality of nucleic acids, or by different vectors of the plurality ofvectors.

Cells Comprising/Expressing the Antigen-Binding Molecules andPolypeptides

The present invention also provides a cell comprising or expressing anantigen-binding molecule, polypeptide or CAR according to the presentinvention. Also provided is a cell comprising or expressing a nucleicacid, a plurality of nucleic acids, a vector or a plurality of vectorsaccording to the invention.

The cell may be a eukaryotic cell, e.g. a mammalian cell. The mammal maybe a primate (rhesus, cynomolgous, non-human primate or human) or anon-human mammal (e.g. rabbit, guinea pig, rat, mouse or other rodent(including any animal in the order Rodentia), cat, dog, pig, sheep,goat, cattle (including cows, e.g. dairy cows, or any animal in theorder Bos), horse (including any animal in the order Equidae), donkey,and non-human primate).

The present invention also provides a method for producing a cellcomprising a nucleic acid(s) or vector(s) according to the presentinvention, comprising introducing a nucleic acid, a plurality of nucleicacids, a vector or a plurality of vectors according to the presentinvention into a cell. In some embodiments, introducing an isolatednucleic acid(s) vector(s) according to the invention into a cellcomprises transformation, transfection, electroporation or transduction(e.g. retroviral transduction).

The present invention also provides a method for producing a cellexpressing/comprising an antigen-binding molecule, polypeptide or CARaccording to the present invention, comprising introducing a nucleicacid, a plurality of nucleic acids, a vector or a plurality of vectorsaccording to the present invention in a cell. In some embodiments, themethods additionally comprise culturing the cell under conditionssuitable for expression of the nucleic acid(s) or vector(s) by the cell.In some embodiments, the methods are performed in vitro.

The present invention also provides cells obtained or obtainable by themethods according to the present invention.

Producing the Antigen-Binding Molecules and Polypeptides

Antigen-binding molecules and polypeptides according to the inventionmay be prepared according to methods for the production of polypeptidesknown to the skilled person.

Polypeptides may be prepared by chemical synthesis, e.g. liquid or solidphase synthesis. For example, peptides/polypeptides can by synthesisedusing the methods described in, for example, Chandrudu et al., Molecules(2013), 18: 4373-4388, which is hereby incorporated by reference in itsentirety.

Alternatively, antigen-binding molecules and polypeptides may beproduced by recombinant expression. Molecular biology techniquessuitable for recombinant production of polypeptides are well known inthe art, such as those set out in Green and Sambrook, Molecular Cloning:A Laboratory Manual (4th Edition), Cold Spring Harbor Press, 2012, andin Nat Methods. (2008); 5(2): 135-146 both of which are herebyincorporated by reference in their entirety. Methods for the recombinantproduction of antigen-binding molecules are also described in Frenzel etal., Front Immunol. (2013); 4: 217 and Kunert and Reinhart, ApplMicrobiol Biotechnol. (2016) 100: 3451-3461, both of which are herebyincorporated by reference in their entirety.

In some cases the antigen-binding molecule of the present invention arecomprised of more than one polypeptide chain. In such cases, productionof the antigen-binding molecules may comprise transcription andtranslation of more than one polypeptide, and subsequent association ofthe polypeptide chains to form the antigen-binding molecule.

For recombinant production according to the invention, any cell suitablefor the expression of polypeptides may be used. The cell may be aprokaryote or eukaryote. In some embodiments the cell is a prokaryoticcell, such as a cell of archaea or bacteria. In some embodiments thebacteria may be Gram-negative bacteria such as bacteria of the familyEnterobacteriaceae, for example Escherichia coli. In some embodiments,the cell is a eukaryotic cell such as a yeast cell, a plant cell, insectcell or a mammalian cell, e.g. CHO, HEK (e.g. HEK293), HeLa or COScells. In some embodiments, the cell is a CHO cell that transiently orstably expresses the polypeptides.

In some cases the cell is not a prokaryotic cell because someprokaryotic cells do not allow for the same folding orpost-translational modifications as eukaryotic cells. In addition, veryhigh expression levels are possible in eukaryotes and proteins can beeasier to purify from eukaryotes using appropriate tags. Specificplasmids may also be utilised which enhance secretion of the proteininto the media.

In some embodiments polypeptides may be prepared by cell-free-proteinsynthesis (CFPS), according using a system described in Zemella et al.Chembiochem (2015) 16(17): 2420-2431, which is hereby incorporated byreference in its entirety.

Production may involve culture or fermentation of a eukaryotic cellmodified to express the polypeptide(s) of interest. The culture orfermentation may be performed in a bioreactor provided with anappropriate supply of nutrients, air/oxygen and/or growth factors.Secreted proteins can be collected by partitioning culturemedia/fermentation broth from the cells, extracting the protein content,and separating individual proteins to isolate secreted polypeptide(s).Culture, fermentation and separation techniques are well known to thoseof skill in the art, and are described, for example, in Green andSambrook, Molecular Cloning; A Laboratory Manual (4th Edition;incorporated by reference herein above).

Bioreactors include one or more vessels in which cells may be cultured.Culture in the bioreactor may occur continuously, with a continuous flowof reactants into, and a continuous flow of cultured cells from, thereactor. Alternatively, the culture may occur in batches. The bioreactormonitors and controls environmental conditions such as pH, oxygen, flowrates into and out of, and agitation within the vessel such that optimumconditions are provided for the cells being cultured.

Following culturing the cells that express the antigen-bindingmolecule/polypeptide(s), the polypeptide(s) of interest may be isolated.Any suitable method for separating proteins from cells known in the artmay be used. In order to isolate the polypeptide it may be necessary toseparate the cells from nutrient medium. If the polypeptide(s) aresecreted from the cells, the cells may be separated by centrifugationfrom the culture media that contains the secreted polypeptide(s) ofinterest. If the polypeptide(s) of interest collect within the cell,protein isolation may comprise centrifugation to separate cells fromcell culture medium, treatment of the cell pellet with a lysis buffer,and cell disruption e.g. by sonification, rapid freeze-thaw or osmoticlysis.

It may then be desirable to isolate the polypeptide(s) of interest fromthe supernatant or culture medium, which may contain other protein andnon-protein components. A common approach to separating proteincomponents from a supernatant or culture medium is by precipitation.Proteins of different solubilities are precipitated at differentconcentrations of precipitating agent such as ammonium sulfate. Forexample, at low concentrations of precipitating agent, water solubleproteins are extracted. Thus, by adding different increasingconcentrations of precipitating agent, proteins of differentsolubilities may be distinguished. Dialysis may be subsequently used toremove ammonium sulfate from the separated proteins.

Other methods for distinguishing different proteins are known in theart, for example ion exchange chromatography and size chromatography.These may be used as an alternative to precipitation, or may beperformed subsequently to precipitation.

Once the polypeptide(s) of interest have been isolated from culture itmay be desired or necessary to concentrate the polypeptide(s). A numberof methods for concentrating proteins are known in the art, such asultrafiltration or lyophilisation.

Compositions

The present invention also provides compositions comprising theantigen-binding molecules, polypeptides, CARs, nucleic acids, expressionvectors and cells described herein.

The antigen-binding molecules, polypeptides, CARs, nucleic acids,expression vectors and cells described herein may be formulated aspharmaceutical compositions or medicaments for clinical use and maycomprise a pharmaceutically acceptable carrier, diluent, excipient oradjuvant. The composition may be formulated for topical, parenteral,systemic, intracavitary, intravenous, intra-arterial, intramuscular,intrathecal, intraocular, intraconjunctival, intratumoral, subcutaneous,intradermal, intrathecal, oral or transdermal routes of administrationwhich may include injection or infusion.

Suitable formulations may comprise the antigen-binding molecule in asterile or isotonic medium. Medicaments and pharmaceutical compositionsmay be formulated in fluid, including gel, form. Fluid formulations maybe formulated for administration by injection or infusion (e.g. viacatheter) to a selected region of the human or animal body.

In some embodiments the composition is formulated for injection orinfusion, e.g. into a blood vessel or tumor.

In accordance with the invention described herein methods are alsoprovided for the production of pharmaceutically useful compositions,such methods of production may comprise one or more steps selected from:producing an antigen-binding molecule, polypeptide, CAR, nucleic acid(or plurality thereof), expression vector (or plurality thereof) or celldescribed herein; isolating an antigen-binding molecule, polypeptide,CAR, nucleic acid (or plurality thereof), expression vector (orplurality thereof) or cell described herein; and/or mixing anantigen-binding molecule, polypeptide, CAR, nucleic acid (or pluralitythereof), expression vector (or plurality thereof) or cell describedherein with a pharmaceutically acceptable carder, adjuvant, excipient ordiluent.

For example, a further aspect the invention described herein relates toa method of formulating or producing a medicament or pharmaceuticalcomposition for use in the treatment of a disease/condition (e.g. acancer), the method comprising formulating a pharmaceutical compositionor medicament by mixing an antigen-binding molecule, polypeptide, CAR,nucleic acid (or plurality thereof), expression vector (or pluralitythereof) or cell described herein with a pharmaceutically acceptablecarrier, adjuvant, excipient or diluent.

Therapeutic and Prophylactic Applications

The antigen-binding molecules, polypeptides, CARs, nucleic acids,expression vectors, cells and compositions described herein find use intherapeutic and prophylactic methods.

The present invention provides an antigen-binding molecule, polypeptide,CAR, nucleic acid (or plurality thereof), expression vector (orplurality thereof), cell or composition described herein for use in amethod of medical treatment or prophylaxis. Also provided is the use ofan antigen-binding molecule, polypeptide, CAR, nucleic acid (orplurality thereof), expression vector (or plurality thereof), cell orcomposition described herein in the manufacture of a medicament fortreating or preventing a disease or condition. Also provided is a methodof treating or preventing a disease or condition, comprisingadministering to a subject a therapeutically or prophylacticallyeffective amount of an antigen-binding molecule, polypeptide, CAR,nucleic acid (or plurality thereof), expression vector (or pluralitythereof), cell or composition described herein.

The methods may be effective to reduce the development or progression ofa disease/condition, alleviation of the symptoms of a disease/conditionor reduction in the pathology of a disease/condition. The methods may beeffective to prevent progression of the disease/condition, e.g. toprevent worsening of, or to slow the rate of development of, thedisease/condition. In some embodiments the methods may lead to animprovement in the disease/condition, e.g. a reduction in the symptomsof the disease/condition or reduction in some other correlate of theseverity/activity of the disease/condition. In some embodiments themethods may prevent development of the disease/condition a later stage(e.g. a chronic stage or metastasis).

It will be appreciated that the articles of the present invention may beused for the treatment/prevention of any disease/condition that wouldderive therapeutic or prophylactic benefit from a reduction in thenumber and/or activity of cells expressing HER3. For example, thedisease/condition may be a disease/condition in which cells expressingHER3 are pathologically implicated, e.g. a disease/condition in which anincreased number/proportion of cells expressing HER3 is positivelyassociated with the onset, development or progression of thedisease/condition, and/or severity of one or more symptoms of thedisease/condition, or for which an increased number/proportion of cellsexpressing HER3, is a risk factor for the onset, development orprogression of the disease/condition.

In some embodiments, the disease/condition to be treated/prevented inaccordance with the present invention is a disease/conditioncharacterised by an increase in the number/proportion/activity of cellsexpressing HER3, e.g. as compared to the number/proportion/activity ofcells expressing HER3 in the absence of the disease/condition.

In some embodiments the disease/condition to be treated/prevented is acancer.

The cancer may be any unwanted cell proliferation (or any diseasemanifesting itself by unwanted cell proliferation), neoplasm or tumor.The cancer may be benign or malignant and may be primary or secondary(metastatic). A neoplasm or tumor may be any abnormal growth orproliferation of cells and may be located in any tissue. The cancer maybe of tissues/cells derived from e.g. the adrenal gland, adrenalmedulla, anus, appendix, bladder, blood, bone, bone marrow, brain,breast, cecum, central nervous system (including or excluding the brain)cerebellum, cervix, colon, duodenum, endometrium, epithelial cells (e.g.renal epithelia), gallbladder, oesophagus, glial cells, heart, ileum,jejunum, kidney, lacrimal gland, larynx, liver, lung, lymph, lymph node,lymphoblast, maxilla, mediastinum, mesentery, myometrium, nasopharynx,omentum, oral cavity, ovary, pancreas, parotid gland, peripheral nervoussystem, peritoneum, pleura, prostate, salivary gland, sigmoid colon,skin, small intestine, soft tissues, spleen, stomach, testis, thymus,thyroid gland, tongue, tonsil, trachea, uterus, vulva, and/or whiteblood cells.

Tumors to be treated may be nervous or non-nervous system tumors.Nervous system tumors may originate either in the central or peripheralnervous system, e.g. glioma, medulloblastoma, meningioma, neurofibroma,ependymoma, Schwannoma, neurofibrosarcoma, astrocytoma andoligodendroglioma. Non-nervous system cancers/tumors may originate inany other non-nervous tissue, examples include melanoma, mesothelioma,lymphoma, myeloma, leukemia, Non-Hodgkin's lymphoma (NHL), Hodgkin'slymphoma, chronic myelogenous leukemia (CML), acute myeloid leukemia(AML), myelodysplastic syndrome (MDS), cutaneous T-cell lymphoma (CTCL),chronic lymphocytic leukemia (CLL), hepatoma, epidermoid carcinoma,prostate carcinoma, breast cancer, lung cancer, colon cancer, ovariancancer, pancreatic cancer, thymic carcinoma, NSCLC, hematologic cancerand sarcoma.

HER3 and its association with and role in cancer is reviewed e.g. inKarachaliou et al., BioDrugs. (2017) 31(1):83-73 and Zhang et al., ActaBiochimica et Biophysica Sinica (2016) 43(1): 39-48, both of which arehereby incorporated by reference in their entirety.

In some embodiments, a cancer is selected from: a cancer comprisingcells expressing HER3, a solid tumor, breast cancer, breast carcinoma,ductal carcinoma, gastric cancer, gastric carcinoma, gastricadenocarcinoma, colorectal cancer, colorectal carcinoma, colorectaladenocarcinoma, head and neck cancer, squamous cell carcinoma of thehead and neck (SCCHN), lung cancer, lung adenocarcinoma, squamous celllung carcinoma, ovarian cancer, ovarian carcinoma, ovarian serousadenocarcinoma, kidney cancer, renal cell carcinoma, renal clear cellcarcinoma, renal cell adenocarcinoma, renal papillary cell carcinoma,pancreatic cancer, pancreatic adenocarcinoma, pancreatic ductaladenocarcinoma, cervical cancer, cervical squamous cell carcinoma, skincancer, melanoma, esophageal cancer, esophageal adenocarcinoma, livercancer, hepatocellular carcinoma, cholangiocarcinoma, uterine cancer,uterine corpus endometrial carcinoma, thyroid cancer, thyroid carcinoma,pheochromocytoma, paraganglioma, bladder cancer, bladder urothelialcarcinoma, prostate cancer, prostate adenocarcinoma, sarcoma andthymoma.

In some embodiments the cancer to be treated in accordance with thepresent invention is selected from: a HER3-expressing cancer, gastriccancer (e.g. gastric carcinoma, gastric adenocarcinoma, gastrointestinaladenocarcinoma), head and neck cancer (e.g. head and neck squamous cellcarcinoma), breast cancer, ovarian cancer (e.g. ovarian carcinoma), lungcancer (e.g. NSCLC, lung adenocarcinoma, squamous lung cell carcinoma),melanoma, prostate cancer, oral cavity cancer (e.g. oropharyngealcancer), renal cancer (e.g. renal cell carcinoma) or colorectal cancer(e.g. colorectal carcinoma), oesophageal cancer, pancreatic cancer, asolid cancer and/or a liquid cancer.

The treatment/prevention may be aimed at one or more of:delaying/preventing the onset/progression of symptoms of the cancer,reducing the severity of symptoms of the cancer, reducing thesurvival/growth/invasion/metastasis of cells of the cancer, reducing thenumber of cells of the cancer and/or increasing survival of the subject.

In some embodiments, the cancer to be treated/prevented comprises cellsexpressing an EGFR family member (e.g. HER3, EGFR, HER2 or HER4), and/orcells expressing a ligand for an EGFR family member. In someembodiments, the cancer to be treated/prevented is a cancer which ispositive for an EGFR family member. In some embodiments, the cancerover-expresses an EGFR family member and/or a ligand for an EGFR familymember. Overexpression of can be determined by detection of a level ofexpression which is greater than the level of expression by equivalentnon-cancerous cells/non-tumor tissue.

Expression may be determined by any suitable means. Expression may begene expression or protein expression. Gene expression can be determinede.g. by detection of mRNA encoding HER3, for example by quantitativereal-time PCR (gRT-PCR). Protein expression can be determined e.g. byfor example by antibody-based methods, for example by western blot,immunohistochemistry, immunocytochemistry, flow cytometry, or ELISA.

In some embodiments, the cancer to be treated/prevented comprises cellsexpressing HER3. In some embodiments, the cancer to be treated/preventedis a cancer which is positive for HER3. In some embodiments, the cancerover-expresses HER3. Overexpression of HER3 can be determined bydetection of a level of expression of HER3 which is greater than thelevel of expression by equivalent non-cancerous cells/non-tumor tissue.

In some embodiments, a patient may be selected for treatment describedherein based on the detection of a cancer expressing HER3, oroverexpressing HER3, e.g. in a sample obtained from the subject.

In some embodiments, the cancer to be treated/prevented comprises cellsexpressing a ligand for HER3 (e.g. NRG1 and/or NRG2). In someembodiments, the cancer to be treated/prevented comprises cellsexpressing a level of expression of NRG1 and/or NRG2 which is greaterthan the level of expression by equivalent non-cancerous cells/non-tumortissue.

Administration of the articles of the present invention is preferably ina “therapeutically effective” or “prophylactically effective” amount,this being sufficient to show therapeutic or prophylactic benefit to thesubject. The actual amount administered, and rate and time-course ofadministration, will depend on the nature and severity of thedisease/condition and the particular article administered. Prescriptionof treatment, e.g. decisions on dosage etc., is within theresponsibility of general practitioners and other medical doctors, andtypically takes account of the disease/disorder to be treated, thecondition of the individual subject, the site of delivery, the method ofadministration and other factors known to practitioners. Examples of thetechniques and protocols mentioned above can be found in Remington'sPharmaceutical Sciences, 20th Edition, 2000, pub. Lippincott, Williams &Wilkins.

Administration may be alone or in combination with other treatments,either simultaneously or sequentially dependent upon the condition to betreated. The antigen-binding molecule or composition described hereinand a therapeutic agent may be administered simultaneously orsequentially.

In some embodiments, the methods comprise additional therapeutic orprophylactic intervention, e.g. for the treatment/prevention of acancer. In some embodiments, the therapeutic or prophylacticintervention is selected from chemotherapy, immunotherapy, radiotherapy,surgery, vaccination and/or hormone therapy. In some embodiments, thetherapeutic or prophylactic intervention comprises leukapheresis. Insome embodiments the therapeutic or prophylactic intervention comprisesa stem cell transplant.

In some embodiments the antigen-binding molecule is administered incombination with an agent capable of inhibiting signalling mediated byan EGFR family member.

Accordingly, the invention provides compositions comprising an articleaccording to the present invention (e.g. an antigen-binding moleculeaccording to the invention) and another agent capable of inhibitingsignalling mediated by an EGFR family member (e.g. EGFR, HER2, HER3 orHER4). Also provided is the use of such compositions in methods ofmedical treatment and prophylaxis of diseases/conditions describedherein.

Also provided are methods for treating/preventing diseases/conditionsdescribed herein comprising administering articles of the presentinvention an article according to the present invention (e.g. anantigen-binding molecule according to the invention) and another agentcapable of inhibiting signalling mediated by an EGFR family member.

Agents capable of inhibiting signalling mediated by EGFR family membersare known in the art, and include e.g. small molecule inhibitor's (e.g.tyrosine kinase inhibitors), monoclonal antibodies (and antigen-bindingfragments thereof), peptide/polypeptide inhibitors (e.g. decoy ligands;receptors or peptide aptamers) and nucleic acids (e.g. antisense nucleicacid, splice-switching nucleic acids or nucleic acid aptamers).Inhibitors of signalling mediated by EGFR family members include agentsthat inhibit signalling through a direct effect on an EGFR familymember, an interaction partner therefore, and/or a downstream factorinvolved in signalling mediated by the EGFR family member.

In some embodiments the antagonist of signalling mediated by an EGFRfamily member inhibits signalling mediated by one or more of EGFR, HER2,HER4 and HER3. Inhibitors of signalling mediated by EGFR family membersare described e.g. in Yamaoka et al., Int. J. Mol. Sci. (2018), 19,3491, which is hereby incorporated by reference in its entirety. In someembodiments the antagonist is a pan-ErbB inhibitor. In some embodimentsthe antagonist is an inhibitor of signalling mediated by EGFR (e.g.cetuximab, panitumumab, gefitinib, erlotinib, lapatinib, afatinib,brigatinib, icotinib, osimertinib, zalutumuniab, vandetanib,necituniumab, nimotuzumab, dacomitinib, duligotuzumab or matuzumab). Insome embodiments the antagonist is an inhibitor of signalling mediatedby HER2 (e.g. trastuzumab, pertuzumab, lapatinib, neratinib, afatinib,dacomitinib, MM-111, MCLA-128 or margetuximab). In some embodiments theantagonist is an inhibitor of signalling mediated by HER3 (e.g.seribantumab, lumretuzumab, elgemtumab, KTN3379, AV-203, GSK2849330,REGN1400, MP-RM-1, EV20, duligotuzumab, MM-111, istiratumab, MCLA-128,patritumab, EZN-3920, RB200 or U3-1402). In some embodiments theantagonist is an inhibitor of signalling mediated by HER4 (e.g.lapatinib, ibrutinib, afatinib, dacomitinib or neratinib).

In some embodiments the antagonist of signalling mediated by an EGFRfamily member inhibits a downstream effector of signalling by an EGFRfamily member. Downstream effectors of signalling by an EGFR familymembers include e.g. PI3K, AKT, KRAS, BRAF, MEK/ERK and mTOR. In someembodiments, the antagonist of signalling mediated by an EGFR familymember is an inhibitor of the MAPK/ERK pathway. In some embodiments, theantagonist of signalling mediated by an EGFR family member is aninhibitor of the PI3K/ATK/mTOR pathway. In some embodiments theantagonist is a PI3K inhibitor (e.g. pictilisib, buparlisib, idelalisib,copanlisib or duvelisib). In some embodiments the antagonist is an AKTinhibitor (e.g. MK-2206, AZD5363, ipatasertib, VQD-002, perifosine ormiltefosine). In some embodiments the antagonist is a BRAF inhibitor(e.g. vemurafenib, dabrafenib, SB590885, XL281, RAF265, encorafenib,GDC-0879, PLX-4720, sorafenib, or LGX818). In some embodiments theantagonist is a MEK/ERK inhibitor (e.g. trametinib, cobimetinib,binimetinib, selumetinib, RD-325901 CI-1040, PD035901, or TAK-733). Insome embodiments the antagonist is a mTOR inhibitor (e.g. rapamycin,deforolimus, temsirolimus, everolimus, ridaforolimus or sapanisertib).

In some embodiments, the cancer to be treated in accordance with anaspect of the present invention (including monotherapy or combinationtherapy) is a cancer which is resistant to treatment with an antagonistof signalling mediated by an EGFR family member (e.g. EGFR, HER2, HER4and/or HER3), e.g. an antagonist as described in the preceding threeparagraphs. In some embodiments the subject to be treated has a cancerwhich is resistant to treatment with an antagonist of signallingmediated by an EGFR family member. In some embodiments the subject to betreated has a cancer which has developed resistance to treatment with anantagonist of signalling mediated by an EGFR family member. In someembodiments the subject to be treated has a cancer which previouslyresponded to treatment with an antagonist of signalling mediated by anEGFR family member, and which is now resistant to treatment with theantagonist. In some embodiments the subject to be treated has a cancerwhich has relapsed and/or progressed following treatment with anantagonist of signalling mediated by an EGFR family member. In someembodiments the subject to be treated has a cancer which initiallyresponded to treatment with an antagonist of signalling mediated by anEGFR family member, but later progressed on said treatment.

The skilled person is readily able to identify cancers and subjectsaccording to the preceding paragraph. Such cancers and subjects may beidentified e.g. through monitoring of the development/progression of thecancer (and/or correlates thereof) over time e.g. during the course oftreatment with an antagonist of signalling mediated by an EGFR familymember. In some embodiments, identification of such subjects/cancers maycomprise analysis of a sample (e.g. a biopsy), e.g. in vitro. In someembodiments the cancer may be determined to comprise cells having amutation which is associated with reduced susceptibility and/orresistance to treatment with the antagonist. In some embodiments thecancer may be determined to comprise cells having upregulated expressionof an EGFR family member.

In particular embodiments, the cancer to be treated is a cancer which isresistant to treatment with an antagonist of signalling mediated by EGFRand/or HER2. In some embodiments the subject to be treated has a cancerwhich is resistant to treatment with an antagonist of signallingmediated by EGFR and/or HER2. In some embodiments the subject to betreated has a cancer which has developed resistance to treatment with anantagonist of signalling mediated by EGFR and/or HER2. In someembodiments the subject to be treated has a cancer which previouslyresponded to treatment with an antagonist of signalling mediated by EGFRand/or HER2, and which is now resistant to treatment with theantagonist. In some embodiments the subject to be treated has a cancerwhich has relapsed and/or progressed following treatment with anantagonist of signalling mediated by EGFR and/or HER2. In someembodiments the subject to be treated has a cancer which initiallyresponded to treatment with an antagonist of signalling mediated by EGFRand/or HER2, but later progressed on said treatment.

In particular embodiments, the cancer to be treated comprises mutationconferring resistance to treatment with an inhibitor of BRAF. In someembodiments, the mutation is mutation at BRAF V600. In some embodiments,the mutation is BRAF V600E or V600K.

In particular embodiments, the cancer to be treated comprises mutationconferring resistance to treatment with an inhibitor of BRAF (e.g.mutation at BRAF V600), and the treatment comprises administration ofvemurafenib or darafenib.

In some embodiments the antigen-binding molecule is administered incombination with an agent capable of inhibiting signalling mediated byan immune checkpoint molecule. In some embodiments the immune checkpointmolecule is e.g. PD-1, CTLA-4, LAG-3, VISTA, TIM-3, TIGIT or BTLA. Insome embodiments the antigen-binding molecule is administered incombination with an agent capable of promoting signalling mediated by acostimulatory receptor. In some embodiments the costimulatory receptoris e.g. CD28, CD80, CD40L, CD86, OX40, 4-1 BB, CD27 or ICOS.

Accordingly, the invention provides compositions comprising an articleaccording to the present invention (e.g. an antigen-binding moleculeaccording to the invention) and an agent capable of inhibitingsignalling mediated by an immune checkpoint molecule. Also provided arecompositions comprising the articles of the present invention and anagent capable of promoting signalling mediated by a costimulatoryreceptor. Also provided is the use of such compositions in methods ofmedical treatment and prophylaxis of diseases/conditions describedherein.

Also provided are methods for treating/preventing diseases/conditionsdescribed herein comprising administering articles of the presentinvention an article according to the present invention (e.g. anantigen-binding molecule according to the invention) and an agentcapable of inhibiting signalling mediated by an immune checkpointmolecule. Also provided are methods for treating/preventingdiseases/conditions described herein comprising administering articlesof the present invention an article according to the present invention(e.g. an antigen-binding molecule according to the intention)) and anagent capable of promoting signalling mediated by a costimulatoryreceptor.

Agents capable of inhibiting signalling mediated by immune checkpointmolecules are known in the art, and include e.g. antibodies capable ofbinding to immune checkpoint molecules or their ligands, and inhibitingsignalling mediated by the immune checkpoint molecule. Other agentscapable of inhibiting signalling mediated by an immune checkpointmolecule include agents capable of reducing gene/protein expression ofthe immune checkpoint molecule or a ligand for the immune checkpointmolecule (e.g. through inhibiting transcription of the gene(s) encodingthe immune checkpoint molecule/ligand, inhibiting post-transcriptionalprocessing of RNA encoding the immune checkpoint molecule/ligand,reducing stability of RNA encoding the immune checkpointmolecule/ligand, promoting degradation of RNA encoding the immunecheckpoint molecule/ligand, inhibiting post-translational processing ofthe immune checkpoint molecule/ligand, reducing stability the immunecheckpoint molecule/ligand, or promoting degradation of the immunecheckpoint molecule/ligand), and small molecule inhibitors.

Agents capable of promoting signalling mediated by costimulatoryreceptors are known in the art, and include e.g. agonist antibodiescapable of binding to costimulatory receptors and triggering orincreasing signalling mediated by the costimulatory receptor. Otheragents capable of promoting signalling mediated by costimulatoryreceptors include agents capable of increasing gene/protein expressionof the costimulatory receptor or a ligand for the costimulatory receptor(e.g. through promoting transcription of the gene(s) encoding thecostimulatory receptor/ligand, promoting post-transcriptional processingof RNA encoding the costimulatory receptor/ligand, increasing stabilityof RNA encoding the costimulatory receptor/ligand, inhibitingdegradation of RNA encoding the costimulatory receptor/ligand, promotingpost-translational processing of the costimulatory receptor/ligand,increasing stability the costimulatory receptor/ligand, or inhibitingdegradation of the costimulatory receptor/ligand), and small moleculeagonists.

In particular embodiments the antigen-binding molecule of the presentinvention is administered in combination with an agent capable ofinhibiting signalling mediated by PD-1. The agent capable of inhibitingsignalling mediated by PD-1 may be a PD-1- or PD-L1-targeted agent. Theagent capable of inhibiting signalling mediated by PD-1 may e.g. be anantibody capable of binding to PD-1 or PD-L1 and inhibitingPD-1-mediated signalling.

In some embodiments, the antigen-binding molecule of the presentinvention is administered in combination with an agent capable ofinhibiting signalling mediated by CTLA-4. The agent capable ofinhibiting signalling mediated by CTLA-4 may be a CTLA-4-targeted agent,or an agent targeted against a ligand for CTLA-4 such as CD80 or CD86.In some embodiments, the agent capable of inhibiting signalling mediatedby CTLA-4 may e.g. be an antibody capable of binding to CTLA-4, CD80 orCD86 and inhibiting CTLA-4-mediated signalling.

In some embodiments, the antigen-binding molecule of the presentinvention is administered in combination with an agent capable ofinhibiting signalling mediated by LAG-3. The agent capable of inhibitingsignalling mediated by LAG-3 may be a LAG-3-targeted agent, or an agenttargeted against a ligand for LAG-3 such as MHC class II. In someembodiments, the agent capable of inhibiting signalling mediated byLAG-3 may e.g. be an antibody capable of binding to LAG-3 or MHC classII and inhibiting LAG-3-mediated signalling.

In some embodiments, the antigen-binding molecule of the presentinvention is administered in combination with an agent capable ofinhibiting signalling mediated by VISTA. The agent capable of inhibitingsignalling mediated by VISTA may be a VISTA-targeted agent, or an agenttargeted against a ligand for VISTA such as VSIG-3 or VSIG-8. In someembodiments, the agent capable of inhibiting signalling mediated byVISTA may e.g. be an antibody capable of binding to VISTA, VS1G-3 orVS1G-8 and inhibiting VISTA-mediated signalling.

In some embodiments, the antigen-binding molecule of the presentinvention is administered in combination with an agent capable ofinhibiting signalling mediated by TIM-3. The agent capable of inhibitingsignalling mediated by TIM-3 may be a TIM-3-targeted agent, or an agenttargeted against a ligand for TIM-3 such as Galectin 9. In someembodiments, the agent capable of inhibiting signalling mediated byTIM-3 may e.g. be an antibody capable of binding to TIM-3 or Galectin 9and inhibiting TIM-3-mediated signalling.

In some embodiments, the antigen-binding molecule of the presentinvention is administered in combination with an agent capable ofinhibiting signalling mediated by TIGIT. The agent capable of inhibitingsignalling mediated by TIGIT may be a TIGIT-targeted agent, or an agenttargeted against a ligand for TIGIT such as CD113, CD112 or CD155. Insome embodiments, the agent capable of inhibiting signalling mediated byTIGIT may e.g. be an antibody capable of binding to TIGIT, CD113, CD112or CD155 and inhibiting TIGIT-mediated signalling.

In some embodiments, the antigen-binding molecule of the presentinvention is administered in combination with an agent capable ofinhibiting signalling mediated by BTLA. The agent capable of inhibitingsignalling mediated by BTLA may be a BTLA-targeted agent, or an agenttargeted against a ligand for BTLA such as HVEM. In some embodiments,the agent capable of inhibiting signalling mediated by BTLA may e.g. bean antibody capable of binding to BTLA or HVEM and inhibitingBTLA-mediated signalling.

In some embodiments methods employing a combination of anantigen-binding molecule of the present invention and an agent capableof inhibiting signalling mediated by an immune checkpoint molecule (e.g.PD-1) provide an improved treatment effect as compared to the effectobserved when either agent is used as a monotherapy. In some embodimentsthe combination of an antigen-binding molecule of the present inventionand an agent capable of inhibiting signalling mediated by an immunecheckpoint molecule (e.g. PD-1) provide a synergistic (i.e.super-additive) treatment effect.

Simultaneous administration refers to administration of theantigen-binding molecule, polypeptide, CAR, nucleic acid (or pluralitythereof), expression vector (or plurality thereof), cell or compositionand therapeutic agent together, for example as a pharmaceuticalcomposition containing both agents (combined preparation), orimmediately after each other and optionally via the same route ofadministration, e.g. to the same artery, vein or other blood vessel.Sequential administration refers to administration of one of theantigen-binding molecule/composition or therapeutic agent followed aftera given time interval by separate administration of the other agent. Itis not required that the two agents are administered by the same route,although this is the case in some embodiments. The time interval may beany time interval.

Chemotherapy and radiotherapy respectively refer to treatment of acancer with a drug or with ionising radiation (e.g. radiotherapy usingX-rays or γ-rays). The drug may be a chemical entity, e.g. smallmolecule pharmaceutical, antibiotic, DNA intercalator, protein inhibitor(e.g. kinase inhibitor), or a biological agent, e.g. antibody, antibodyfragment, aptamer, nucleic acid (e.g. DNA, RNA), peptide, polypeptide,or protein. The drug may be formulated as a pharmaceutical compositionor medicament. The formulation may comprise one or more drugs (e.g. oneor more active agents) together with one or more pharmaceuticallyacceptable diluents, excipients or carriers.

A treatment may involve administration of more than one drug. A drug maybe administered alone or in combination with other treatments, eithersimultaneously or sequentially dependent upon the condition to betreated. For example, the chemotherapy may be a co-therapy involvingadministration of two drugs, one or more of which may be intended totreat the cancer.

The chemotherapy may be administered by one or more routes ofadministration, e.g. parenteral, intravenous injection, oral,subcutaneous, intradermal or intratumoral.

The chemotherapy may be administered according to a treatment regime.The treatment regime may be a pre-determined timetable, plan, scheme orschedule of chemotherapy administration which may be prepared by aphysician or medical practitioner and may be tailored to suit thepatient requiring treatment. The treatment regime may indicate one ormore of: the type of chemotherapy to administer to the patient; the doseof each drug or radiation; the time interval between administrations;the length of each treatment; the number and nature of any treatmentholidays, if any etc. For a co-therapy a single treatment regime may beprovided which indicates how each drug is to be administered.

Chemotherapeutic drugs may be selected from; Abemaciclib, AbirateroneAcetate, Abitrexate (Methotrexate), Abraxane (PaclitaxelAlbumin-stabilized Nanoparticle Formulation), ABVD, ABVE, ABVE-PC, AC,Acalabrutinib, AC-T, Adcetris (Brentuximab Vedotin), ADE,Ado-Trastuzumab Emtansine, Adriamycin (Doxorubicin Hydrochloride),Afatinib Dimaleate, Afinitor (Everolimus), Akynzeo (Netupitant andPalonosetron Hydrochloride), Aldara (Imiquimod), Aldesleukin, Alecensa(Alectinib), Alectinib, Alemluzumab, Alimta (Pemetrexed Disodium),Aliqopa (Copanlisib Hydrochloride), Alkeran for Injection (MelphalanHydrochloride), Alkeran Tablets (Melphalan), Aloxi (PalonosetronHydrochloride), Alunbrig (Brigatinib), Ambochiorin (Chlorambucil),Ambociorin (Chlorambucil), Amifostine, Aminolevulinic Acid, Anastrozole,Aprepitant, Aredia (Pamidronate Disodium), Arimidex (Anastrozole),Aromasin (Exemestane), Arranon (Nelarabine), Arsenic Trioxide, Arzerra(Ofatumumab), Asparaginase Erwinia chrysanthemi, Atezolizumab, Avastin(Bevacizumab), Avelumab, Axicabtagene Ciloleucel, Axitinib, Azacitidine,Bavencio (Ayelumab), BEACOPP, Becenum (Carmustine), Beleodag(Belinostat), Belinostat, Bendamustine Hydrochloride, BEP, Besponsa(Inotuzumab Ozogamicin), Bevacizumab, Bexarotene, Bexxar (Tositumomaband Iodine I 131 Tositumomab), Bicalutamide, BiCNU (Carmustine),Bleomycin, Blinatumomab, Blincyto (Blinatumomab), Bortezomib, Bosulif(Bosutinib), Bosutinib, Brentuximab Vedotin, Brigatinib, BuMel,Busulfan, Busulfex (Busulfan), Cabazitaxel, Cabometyx(Cabozantinib-S-Malate), Cabozantinib-S-Malate, CAF, Calquence(Acalabrutinib), Campath (Alemluzumab), Camptosar (IrinotecanHydrochloride), Capecitabine, CAPOX, Carac (Fluorouracil—Topical),Carboplatin, CARBOPLATIN-TAXOL, Carfilzomib, Carmubris (Carmustine),Carmustine, Carmustine Implant, Casodex (Bicalutamide), CEM, Ceritinib,Cerubidine (Daunorubicin Hydrochloride), Cervarix (Recombinant HPVBivalent Vaccine), Cetuximab, CEV, Chlorambucil,CHLORAMBUCIL-PREDNISONE, CHOP, Cisplatin, Cladribine, Clafen(Cyclophosphamide), Clofarabine, Clofarex (Clofarabine), Clolar(Clofarabine), CMF, Cobimetinib, Cometriq (Cabozantinib-S-Malate),Copanlisib Hydrochloride, COPDAC, COPP, COPP-ABV, Cosmegen(Dactinomycin), Cotellic (Cobimetinib), Crizotinib, CVP,Cyclophosphamide, Cyfos (Ifosfamide), Cyramza (Ramucirumab), Cytarabine,Cytarabine Liposome, Cytosar-U (Cytarabine), Cytoxan (Cyclophosphamide),Dabrafenib, Dacarbazine, Dacogen (Decitabine), Dactinomycin,Daratumumab, Darzalex (Daratumumab), Dasatinib, DaunorubicinHydrochloride, Daunorubicin Hydrochloride and Cytarabine Liposome,Decitabine, Defibrotide Sodium, Defitelio (Defibrotide Sodium),Degarelix, Denileukin Diftitox, Denosumab, DepoCyt (CytarabineLiposome), Dexamethasone, Dexrazoxane Hydrochloride, Dinutuximab,Docetaxel, Doxil (Doxorubicin Hydrochloride Liposome), DoxorubicinHydrochloride, Doxorubicin Hydrochloride Liposome, Dox-SL (DoxorubicinHydrochloride Liposome), DTIC-Dome (Dacarbazine), Durvalumab, Efudex(Fluorouracil—Topical), Elitek (Rasburicase), Ellence (EpirubicinHydrochloride), Elotuzumab, Eloxatin (Oxaliplatin), Eltrombopag Olamine,Emend (Aprepitant), Empliciti (Elotuzumab), Enasidenib Mesylate,Enzalutamide, Epirubicin Hydrochloride, EPOCH, Erbitux (Cetuximab),Eribulin Mesylate, Erivedge (Vismodegib), Erlotinib Hydrochloride,Erwinaze (Asparaginase Erwinia chrysanthemi), Ethyol (Amifostine),Etopophos (Etoposide Phosphate), Etoposide, Etoposide Phosphate, Evacet(Doxorubicin Hydrochloride Liposome), Everolimus, Evista (RaloxifeneHydrochloride), Evomela (Melphalan Hydrochloride), Exemestane, 5-FU(Fluorouracil Injection), 5-FU (Fluorouracil—Topical), Fareston(Toremifene), Farydak (Panobinostat), Faslodex (Fulvestrant), FEC,Femara (Letrozole), Filgrastim, Fludara (Fiudarabine Phosphate),Fludarabine Phosphate, Fluoroplex (Fluorouracil—Topical), Fluorouracilinjection, Fluorouracil—Topical, Flutamide, Folex (Methotrexate), FolexPFS (Methotrexate), FOLFIRI, FOLFIRI-BEVACIZUMAB, FOLFIRI-CETUXIMAB,FOLFIRINOX, FOLFOX, Folotyn (Pralatrexate), FU-LV, Fulvestrant, Gardasil(Recombinant HPV Quadrivalent Vaccine), Gardasil 9 (Recombinant HPVNonavalent Vaccine), Gazyva (Obinutuzumab), Gefitinib, GemcitabineHydrochloride, GEMCITABINE-CISPLATIN, GEMCITABINE-OXALIPLATIN,Gemtuzumab Ozogamicin, Gemzar (Gemcitabine Hydrochloride), Gilotrif(Afatinib Dimaleate), Gleevec (Imatinib Mesylate), Gliadel (CarmustineImplant), Gliadel wafer (Carmustine Implant), Glucarpidase, GoserelinAcetate, Halaven (Eribulin Mesylate), Hemangeol (PropranololHydrochloride), Herceptin (Trastuzumab), HPV Bivalent Vaccine,Recombinant, HPV Nonavalent Vaccine, Recombinant, HPV QuadrivalentVaccine, Recombinant, Hycamtin (Topotecan Hydrochloride), Hydrea(Hydroxyurea), Hydroxyurea, Hyper-CVAD, Ibrance (Palbociclib),Ibritumomab Tiuxetan, Ibrutinib, ICE, Iclusig (Ponatinib Hydrochloride),Idamycin (Idarubicin Hydrochloride), Idarubicin Hydrochloride,Idelalisib, Idhifa (Enasidenib Mesylate), Ifex (Ifosfamide), Ifosfamide,Ifosfamidum (Ifosfamide), IL-2 (Aldesleukin), Imatinib Mesylate,Imbruvica (Ibrutinib), Imfinzi (Durvalumab), Imiquimod, Imlygic(Talimogene Laherparepvec), Inlyta (Axitinib), Inotuzumab Ozogamicin,Interferon Alfa-2b, Recombinant, Interleukin-2 (Aldesleukin), Intron A(Recombinant Interferon Alfa-2b), Iodine I 131 Tositumomab andTositumomab, Ipilimumab, Iressa (Gefitinib), Irinotecan Hydrochloride,Irinotecan Hydrochloride Liposome, Istodax (Romidepsin), Ixabepilone,Ixazomib Citrate, Ixempra (Ixabepilone), Jakafi (Ruxolitinib Phosphate),JEB, Jevtana (Cabazitaxel), Kadcyla (Ado-Trastuzumab Emtansine),Keoxifene (Raloxifene Hydrochloride), Kepivance (Palifermin), Keytruda(Pembrolizumab), Kisqali (Ribociclib), Kymriah (Tisagenlecleucel),Kyprolis (Carfilzomib), Lanreotide Acetate, Lapatinib Ditosylate,Lartruvo (Olaratumab), Lenalidomide, Lenvatinib Mesylate, Lenvima(Lenvatinib Mesylate), Letrozole, Leucovorin Calcium, Leukeran(Chlorambucil), Leuprolide Acetate, Leustatin (Cladribine), Levulan(Aminolevulinic Acid), Linfolizin (Chlorambucil), LipoDox (DoxorubicinHydrochloride Liposome), Lomustine, Lonsurf (Trifluridine and TipiracilHydrochloride), Lupron (Leuprolide Acetate), Lupron Depot (LeuprolideAcetate), Lupron Depot-Ped (Leuprolide Acetate), Lynparza (Olaparib),Marqibo (Vincristine Sulfate Liposome), Matulane (ProcarbazineHydrochloride), Mechlorethamine Hydrochloride, Megestrol Acetate,Mekinist (Trametinib), Melphalan, Melphalan Hydrochloride,Mercaptopurine, Mesna, Mesnex (Mesna), Methazolastone (Temozolomide),Methotrexate, Methotrexate LPF (Methotrexate), Methylnaltrexone Bromide,Mexate (Methotrexate), Mexate-AQ (Methotrexate), Midostaurin, MitomycinC, Mitoxantrone Hydrochloride, Mitozytrex (Mitomycin C), MOPP, Mozobil(Plerixafor), Mustargen (Mechlorethamine Hydrochloride), Mutamycin(Mitomycin C), Myleran (Busulfan), Mylosar (Azacitidine), Mylotarg(Gemtuzumab Ozogamicin), Nanoparticle Paclitaxel (PaclitaxelAlbumin-stabilized Nanoparticle Formulation), Navelbine (VinorelbineTartrate), Necitumumab, Nelarabine, Neosar (Cyclophosphamide), NeratinibMaleate, Nerlynx (Neratinib Maleate), Netupitant and PalonosetronHydrochloride, Neulasta (Pegfilgrastim), Neupogen (Filgrastim), Nexavar(Sorafenib Tosylate), Nilandron (Nilutamide), Nilotinib, Nilutamide,Ninlaro (Ixazomib Citrate), Niraparib Tosylate Monohydrate, Nivolumab,Nolvadex (Tamoxifen Citrate), Nplate (Romiplostim), Obinutuzumab, Odomzo(Sonidegib), OEPA, Ofatumumab, OFF, Olaparib, Olaratumab, OmacetaxineMepesuccinate, Oncaspar (Pegaspargase), Ondansetron Hydrochloride,Onivyde (Irinotecan Hydrochloride Liposome), Ontak (DenileukinDiftitox), Opdivo (Nivolumab), OPPA, Osimertinib, Oxaliplatin,Paclitaxel, Paclitaxel Albumin-stabilized Nanoparticle Formulation, PAD,Palbociclib, Palifermin, Palonosetron Hydrochloride, PalonosetronHydrochloride and Netupitant, Pamidronate Disodium, Panitumumab,Panobinostat, Paraplat (Carboplatin), Paraplatin (Carboplatin),Pazopanib Hydrochloride, PCV, PEB, Pegaspargase, Pegfilgrastim,Peginterferon Alfa-2b, PEG-Intron (Peginterferon Alfa-2b),Pembrolizumab, Pemetrexed Disodium, Perjeta (Pertuzumab), Pertuzumab,Platinol (Cisplatin), Platinol-AQ (Cisplatin), Plerixafor, Pomalidomide,Pomalyst (Pomalidomide), Ponatinib Hydrochloride, Portrazza(Necitumumab), Pralatrexate, Prednisone, Procarbazine Hydrochloride,Proleukin (Aldesleukin), Prolia (Denosumab), Promacta (EltrombopagOlamine), Propranolol Hydrochloride, Provenge (Sipuleucel-T), Purinethol(Mercaptopurine), Purixan (Mercaptopurine), [No Entries], Radium 223Dichloride, Raloxifene Hydrochloride, Ramucirumab, Rasburicase, R-CHOP,R-CVP, Recombinant Human Papillomavirus (HPV) Bivalent Vaccine,Recombinant Human Papillomavirus (HPV) Nonavalent Vaccine, RecombinantHuman Papillomavirus (HPV) Quadrivalent Vaccine, Recombinant InterferonAlfa-2b, Regorafenib, Relistor (Methylnaltrexone Bromide), R-EPOCH,Revlimid (Lenalidomide), Rheumatrex (Methotrexate), Ribociclib, R-ICE,Rituxan (Rituximab), Rituxan Hycela (Rituximab and Hyaluronidase Human),Rituximab, Rituximab and Hyaluronidase Human, Rolapitant Hydrochloride,Romidepsin, Romiplostim, Rubidomycin (Daunorubicin Hydrochloride),Rubraca (Rucaparib Camsylate), Rucaparib Camsylate, RuxolitinibPhosphate, Rydapt (Midostaurin), Sclerosol Intrapleural Aerosol (Talc),Siltuximab, Sipuleucel-T, Somatuline Depot (Lanreotide Acetate),Sonidegib, Sorafenib Tosylate, Sprycel (Dasatinib), STANFORD V, SterileTalc Powder (Talc), Steritalc (Talc), Stivarga (Regorafenib), SunitinibMalate, Sutent (Sunitinib Malate), Sylatron (Peginterferon Alfa-2b),Sylvant (Siltuximab), Synribo (Omacetaxine Mepesuccinate), Tabloid(Thioguanine), TAC, Tafinlar (Dabrafenib), Tagrisso (Osimertinib), Talc,Talimogene Lahergarepvec, Tamoxifen Citrate, Tarabine PFS (Cytarabine),Tarceva (Erlotinib Hydrochloride), Targretin (Bexarotene), Tasigna(Nilotinib), Taxol (Paclitaxel), Taxotere (Docetaxel), Tecentriq(Atezolizumab), Temodar (Temozolomide), Temozolomide, Temsirolimus,Thalidomide, Thalomid (Thalidomide), Thioguanine, Thiotepa,Tisagenlecieucel, Tolak (Fluorouracil—Topical), Topotecan Hydrochloride,Toremifene, Torisel (Temsirolimus), Tositumomab and Iodine I 131Tositumomab, Totect (Dexrazoxane Hydrochloride), TPF, Trabectedin,Trametinib, Trastuzumab, Treanda (Bendamustine Hydrochloride),Trifluridine and Tipiracil Hydrochloride, Trisenox (Arsenic Trioxide),Tykerb (Lapatinib Ditosylate), Unituxin (Dinutuximab), UridineTriacetate, VAC, Valrubicin, Valstar (Valrubicin), Vandetanib, VAMP,Varubi (Rolapitant Hydrochloride), Vectibix (Panitumumab), VeIP, Velban(Vinblastine Sulfate), Velcade (Bortezomib), Velsar (VinblastineSulfate), Vemurafenib, Venclexta (Venetoclax), Venetoclax, Verzenio(Abemaciclib), Viadur (Leuprolide Acetate), Vidaza (Azacitidine),Vinblastine Sulfate, Vincasar PFS (Vincristine Sulfate), VincristineSulfate, Vincristine Sulfate Liposome, Vinorelbine Tartrate, VIP,Vismodegib, Vistogard (Uridine Triacetate), Voraxaze (Glucarpidase),Vorinostat, Votrient (Pazopanib Hydrochloride), Vyxeos (DaunorubicinHydrochloride and Cytarabine Liposome), Wellcovorin (LeucovorinCalcium), Xaikori (Crizotinib), Xeloda (Capecitabine), XELIRI, XELOX,Xgeva (Denosumab), Xofigo (Radium 223 Dichloride), Xtandi(Enzalutamide), Yervoy (Ipilimumab), Yescarta (Axicabtagene Ciloleucel),Yondelis (Trabectedin), Zaltrap (Ziv-Aflibercept), Zarxio (Filgrastim),Zejula (Niraparib Tosylate Monohydrate), Zelboraf (Vemurafenib), Zevalin(Ibritumomab Tiuxetan), Zinecard (Dexrazoxane Hydrochloride),Ziv-Aflibercept, Zofran (Ondansetron Hydrochloride), Zoladex (GoserelinAcetate), Zoledronic Acid, Zolinza (Vorinostat), Zometa (ZoledronicAcid), Zydelig (Idelalisib), Zykadia (Ceritinib) and Zytiga (AbirateroneAcetate).

In some embodiments the antigen-binding molecule of the invention isadministered in combination with one or more of: trastuzumab, cetuximab,cisplatin, 5-FU or capecitabine. In some embodiments the antigen-bindingmolecule of the invention is administered in combination withtrastuzumab and cisplatin, and 5-FU or capecitabine.

In some embodiments the antigen-binding molecule of the invention isadministered in combination with cetuximab. Administration incombination with cetuximab is contemplated in particular for thetreatment of head and neck cancer (e.g. head and neck squamous cellcarcinoma).

Multiple doses of the antigen-binding molecule, polypeptide, CAR,nucleic acid (or plurality thereof), expression vector (or pluralitythereof), cell or composition may be provided. One or more, or each, ofthe doses may be accompanied by simultaneous or sequentialadministration of another therapeutic agent.

Multiple doses may be separated by a predetermined time interval, whichmay be selected to be one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or31 days, or 1, 2, 3, 4, 5, or 6 months. By way of example, doses may begiven once every 7, 14, 21 or 28 days (plus or minus 3, 2, or 1 days).

Methods of Detection

The invention also provides the articles of the present invention foruse in methods for detecting, localizing or imaging HER3, or cellsexpressing HER3.

The antigen-binding molecules described herein may be used in methodsthat involve the antigen-binding molecule to HER3. Such methods mayinvolve detection of the bound complex of the antigen-binding moleculeand HER3.

As such, a method is provided, comprising contacting a samplecontaining, or suspected to contain, HER3, and detecting the formationof a complex of the antigen-binding molecule and HER3. Also provided isa method comprising contacting a sample containing, or suspected tocontain, a cell expressing HER3, and detecting the formation of acomplex of the antigen-binding molecule and a cell expressing HER3.

Suitable method formats are well known in the art, includingimmunoassays such as sandwich assays, e.g. ELISA. The methods mayinvolve labelling the antigen-binding molecule, or target(s), or both,with a detectable moiety, e.g. a fluorescent label, phosphorescentlabel, luminescent label, immuno-detectable label, radiolabel, chemical,nucleic acid or enzymatic label as described herein. Detectiontechniques are well known to those of skill in the art and can beselected to correspond with the labelling agent.

Methods of this kind may provide the basis of methods for the diagnosticand/or prognostic evaluation of a disease or condition, e.g. a cancer.Such methods may be performed in vitro on a patient sample, or followingprocessing of a patient sample. Once the sample is collected, thepatient is not required to be present for the in vitro method to beperformed, and therefore the method may be one which is not practised onthe human or animal body. In some embodiments the method is performed invivo.

Detection in a sample may be used for the purpose of diagnosis of adisease/condition (e.g. a cancer), predisposition to adisease/condition, or for providing a prognosis (prognosticating) for adisease/condition, e.g. a disease/condition described herein. Thediagnosis or prognosis may relate to an existing (previously diagnosed)disease/condition.

Such methods may involve detecting or quantifying HER3 or cellsexpressing HER3, e.g. in a patient sample. Where the method comprisesquantifying the relevant factor, the method may further comprisecomparing the determined amount against a standard or reference value aspart of the diagnostic or prognostic evaluation. Otherdiagnostic/prognostic tests may be used in conjunction with thosedescribed herein to enhance the accuracy of the diagnosis or prognosisor to confirm a result obtained by using the tests described herein.

A sample may be taken from any tissue or bodily fluid. The sample maycomprise or may be derived from: a quantity of blood; a quantity ofserum derived from the individual's blood which may comprise the fluidportion of the blood obtained after removal of the fibrin dot and bloodcells; a tissue sample or biopsy; pleural fluid; cerebrospinal fluid(CSF); or cells isolated from said individual. In some embodiments, thesample may be obtained or derived from a tissue or tissues which areaffected by the disease/condition (e.g. tissue or tissues in whichsymptoms of the disease manifest, or which are involved in thepathogenesis of the disease/condition).

The present invention also provides methods for selecting/stratifying asubject for treatment with a HER3-targeted agent. In some embodiments asubject is selected for treatment/prevention in accordance with theinvention, or is identified as a subject which would benefit from suchtreatment/prevention, based on detection/quantification of HER3, orcells expressing HER3, e.g. in a sample obtained from the individual.

Subjects

The subject in accordance with aspects the invention described hereinmay be any animal or human. The subject is preferably mammalian, morepreferably human. The subject may be a non-human mammal, but is morepreferably human. The subject may be male or female. The subject may bea patient. A subject may have been diagnosed with a disease or conditionrequiring treatment (e.g. a cancer), may be suspected of having such adisease/condition, or may be at risk of developing/contracting such adisease/condition.

In embodiments according to the present invention the subject ispreferably a human subject. In some embodiments, the subject to betreated according to a therapeutic; or prophylactic method of theinvention herein is a subject having, or at risk of developing, acancer. In embodiments according to the present invention, a subject maybe selected for treatment according to the methods based oncharacterisation for certain markers of such disease/condition.

Kits

In some aspects of the invention described herein a kit of parts isprovided. In some embodiments the kit may have at least one containerhaving a predetermined quantity of an antigen-binding molecule,polypeptide, CAR, nucleic acid (or plurality thereof), expression vector(or plurality thereof), cell or composition described herein.

In some embodiments, the kit may comprise materials for producing anantigen-binding molecule, polypeptide, CAR, nucleic acid (or pluralitythereof), expression vector (or plurality thereof), cell or compositiondescribed herein.

The kit may provide the antigen-binding molecule, polypeptide, CAR,nucleic acid (or plurality thereof), expression vector (or pluralitythereof), cell or composition together with instructions foradministration to a patient in order to treat a specifieddisease/condition.

In some embodiments the kit may further comprise at least one containerhaving a predetermined quantity of another therapeutic agent (e.g.anti-infective agent or chemotherapy agent). In such embodiments, thekit may also comprise a second medicament or pharmaceutical compositionsuch that the two medicaments or pharmaceutical compositions may beadministered simultaneously or separately such that they provide acombined treatment for the specific disease or condition. Thetherapeutic agent may also be formulated so as to be suitable forinjection or infusion to a tumor or to the blood.

Sequence Identity

As used herein, “sequence identity” refers to the percent ofnucleotides/amino acid residues in a subject sequence that are identicalto nucleotides/amino acid residues in a reference sequence, afteraligning the sequences and, if necessary, introducing gaps, to achievethe maximum percent sequence identity between the sequences. Pairwiseand multiple sequence alignment for the purposes of determining percentsequence identity between two or more amino acid or nucleic acidsequences can be achieved in various ways known to a person of skill inthe art, for instance, using publicly available computer software suchas ClustalOmega (Boding, J. 2005, Bioinformatics 21, 951-960), T-coffee(Notredame et al. 2000, J. Mol. Biol. (2000) 302, 205-217), Kalign(Lassmann and Sonnhammer 2005, BMC Bioinformatics, 6(298)) and MAFFT(Katoh and Standley 2013, Molecular Biology and Evolution, 30(4) 772-780software. When using such software, the default parameters, e.g. for gappenalty and extension penalty, are preferably used.

Sequences SEQ ID NO: DESCRIPTION SEQUENCE 1 Human HER3MRANDALQVLGLLFSLARGSEVGNSQAVCPGTLNGLSVTGDAENQYQTLYKLYERCEVVMGNLEisoform 1 (UniProt:IVLTGHNADLSFLQWIREVTGYVLVAMNEFSTLPLPNLRVVRGTQVDGKFAIFVMLNYNTNSSHAP21860-1, v1)LRQLRLTQLTEILSGGVYIEKNDKLCHMDTIDWRDIVRDRDAEIVVKDNGRSCPPCHEVCKGRCWGPGSEDCQTLTKTICAPQCNGHCFGPNPNQCCHDECAGGCSGPQDTDCFACRHFNDSGACVPRCPQPLVYNKLTCLEPNPHTKYQYGGVCVASCPHNFVVDQTSCVRACPPDKMEVDKNGLKMCEPCGGLCPKACEGTGSGSRFQTVDSSNIDGFVNCTKILGNLDFLITGLNGDPWHKIPALDPEKLNVFRTVREITGYLNIQSWPPHMHNFSVFSNLTTIGGRSLYNRGFSLLIMKNLNVTSLGFRSLKEISAGRIYISANRQLCYHHSLNWTKVLRGPTEERLDIKHNRPRRDCVAEGKVCDPLCSSGGCWGRGPGQCLSCRNYSRGGVCVTHCNFLNGEPREFAHEAECFSCHPECQPMEGTATCNGSGSDTCAQCAHFRDGPHCVSSCPHGVLGAKGPIYKYPDVQNECRPCHENCTQGCKGPELQDCLGQTLVLIGKTHLTMALTVIAGLVVIFMMLGGTFLYWRGRRIQNKRAMRRYLERGESIEPLDPSEKANKVLARIFKETELRKLKVLGSGVFGTVHKGVWIPEGESIKIPVCIKVIEDKSGRQSFQAVTDHMLAIGSLDHAHIVRLLGLCPGSSLQLVTQYLPLGSLLDHVRQHRGALGPQLLLNWGVQIAKGMYYLEEHGMVHRNLAARNVLLKSPSQVQVADFGVADLLPPDDKQLLYSEAKTPIKWMALESIHFGKYTHQSDVWSYGVTVWELMTFGAEPYAGLRLAEVPDLLEKGERLAQPQICTIDVYMVMVKCWMIDENIRPTFKELANEFTRMARDPPRYLVIKRESGPGIAPGPEPHGLTNKKLEEVELEPELDLDLDLEAEEDNLATTTLGSALSLPVGTLNRPRGSQSLLSPSSGYMPMVQGNLGESCQESAVSGSSERCPRPVSLHPMPRGCLASESSEGHVTGSEAELQEKVSMCRSRSRSRSPRPRGDSAYHSQRHSLLTPVTPLSPPGLEEEDVNGYVMPDTHLKGTPSSREGTLSSVGLSSVLGTEEEDEDEEYEYMNRRRRHSPPHPPRPSSLEELGYEYMDVGSDLSASLGSTQSCPLHPVPIMPTAGTTPDEDYEYMNRQRDGGGPGGDYAMAGACPASEQGYEEMRAFQGPGHQAPHVHYARLKTLRSLEATDSAFDNPDYWHSRLFPKANAQRT 2 Human HER3MRANDALQVLGLLFSLARGSEVGNSQAVCPGTLNGLSVTGDAENQYQTLYKLYERCEVVMGNLEisoform 2 (UniProt:IVLTGHNADLSFLQWIREVTGYVLVAMNEFSTLPLPNLRVVRGTQWDGKFAIFVMLNYNTNSSHAP21860-2) LRQLRLTQLTGQFPMVPSGLTPQPAQDWYLLDDDPRLLTLSASSKVPVTLAAV 3Human HER3MRANDALQVLGLLFSLARGSEVGNSQAVCPGTLNGLSVTGDAENGYQTLYKLYERCEVVMGNLEisoform 3 (UnniProt:IVLTGHNADLSFLQWIREVTGYVLVAMNEFSTLPLPNLRVVRGTQVYDGKFAIFVMLNYNTNSSHAP21860-3)LRQLRLTQLTEILSGGVYIEKNDKLCHMDTIDWRDIVRDRDAEIVVKDNGRSCPPCHEVCKGRCWGPGSEDCQTLTKTICAPQCNGHCFGPNPNQCCHDECAGGCSGPQDTDCFACRHFNDSGACVPRCPQPLVYNKLTFQLERNPHTKYQYGGVCVASCPHNFVVDQTSCVRACPPDKMEVDKNGLKMCEPCGGLCPKAF 4 Human HER3MGNLEIVLTGHNADLSFLQWIREVTGYVLVAMNEFSTLPLPNLRVVRGTQVYDGKFAIFVMLNYNTisoform 4 (UniProt:NSSHALRQLRLTQLTEILSGGVYIEKNDKLCHMDTIDWRDIVRDRDAEIVVKDNGRSCPPCHEVCKP21860-4 GRCWGPGSEDCQTLTKTICAPQCNGHCFGPNPNQCCHDECAGGCSGPQDTDCFACRHFNDSGACVPRCPQRLVYNKLTFQLEPNPHTKYQYGGVCVASCPHNFVVDQTSCVRACPPDKMEVDKNGLKMCEPCGGLCPKACEGTGSGSRFQTVDSSNIDGFVNCTKILGNLDFLITGLNGDPWHKIPALDPEKLNVFRTVREITGYLNIQSWPPHMHNFSVFSNLTTIGGRSLYNRGFSLLIMKNLNVTSLGFRSLKEISAGRIYISANRQLCYHHSLNWTKVLRGPTEERLDIKHNRPRRDCVAEGKVCDPLCSSGGCWGPGPGQCLSCRNYSRGGVCVTHCNFLNGEPREFAHEAECFSCHPECQPMEGTATCNGSGSDTCAQCAHFRDGPHCVSSCPHGVLGAKGPIYKYPDVQNECRPCHENCTQGCKGPELQDCLGQTLVLIGKTHLTMALTVIAGLVVIFMMLGGTFLYWRGRRIQNKRAMRRYLERGESIEPLDPSEKANKVLARIFKETELRKLKVLGSGVFGTHKGVWIPEGESIKIPVCIKVIEDKSGRQSFQAVTDHMLAIGLDHAHIVRLLGLCPGSSLQLVTQYLPLGSLLDHVRQHRGALGPQLLLNWGVQIAKGMYYLEEHGMVHRNLAARNVLLKSPSQVQVADFGVADLLPPDDKQLLYSEAKTPIKWMALESIHFGKYTHQSDVWSYGVTVWELMTFGAEPYAGLRLAEVPDLLEKGERLAQPQICTIDVYMVMVKCWMIDENIRPTFKELANEFTRMARDPPRYLVIKRESGPGIAPGREPHGLTNKKLEEVELEPELDLDLDLEAEEDNLATTTLGSALSLPVGTLNRPRGSQSLLSPSSGYMPMNQGNLGESCQESAVSGSSERCPRPVSLHPMPRGCLASESSEGHVTGSEAELQEKVSMCRSRSRSRSPRPRGDSAYHSQRHSLLTPVTPLSPPGLEEEDVNGYVMPDTHLKGTPSSREGTLSSVGLSSVLGTEEEDEDEEYEYMNRRRRHSPPHPPRPSSLEELGYEYMDVGSDLSASLGSTQSCPLHPVPIMPTAGTTPDEDYEYMNRQRDGGGPGGDYAAMGACPASEQGYEEMRAFQGPGHQAPHVHYARLKTLRSLEATDSAFDNPDYWHSRLFPKANAQRT 5 Human HER3MALTVIAGLVVIFMMLGGTFLYWRGRRIQNKRAMRRYLERGESIEPLDPSEKANKVLARIFKETELisoform 5 (UniProt:RKLKVLGSGVFGTVHKGVWIPEGESIKIPVCIKVIEDKSGRQSFQAVTDHMLAIGSLDHAHIVRLLGP21860-5)LCPGSSLQLVTQYLPLGSLLDHVRQHRGALGPQLLLNWGVQIAKGMYYLEEHGMVHRNLAARNVLLKSPSQVQVADFGVADLLPPDDKQLLYSEAKTPIKWMALESIHFGKYTHQSDVWSYGVTVWELMTFGAERYAGLRLAEVPDLLEKGERLAQPQICTIDVYMVMVKCWMIDENIRPTFKELANEFTRMARDPPRYLVIKRESGPGIAPGPEPHGLTNKKLEEVELEPELDLDLDLEAEEDNLATTTLGSALSLPVGTLNRPRGSQSLLSPSSGYMPMNQGNLGESCQESAVSGSSERCPRPVSLHPMPRGCLASESSEGHVTGSEAELQEKVSMCRSRSRSRSPRPRGDSAYHSQPHSLLTPVTPLSPPGLEEEDVNGYVMPDTHLKGTPSSREGTLSSVGLSSVLGTEEEDEDEEYEYMNRRRPHSPPHPPRPSSLEELGYEYMDVGSDLSASLGSTQSCPLHPVPIMPTAGTTPDEDYEYMNPQRDGGGPGGDYAAMGACPASEQGYEEMRAFQGPGHQAPHVHYARLKTLRSLEATDSAFDNPDYWHSRLFPKANAQRT 6 Mature humanSEVGNSQAVCPGTLNGLSVTGDAENQYQTLYKLYERCEVVMGNLEIVLTGHNADLSFLQWIREVTHER3 isoform 1GYVLVAMNEFSTLPLPNLRVVRGTQVYDGKFAIFVMLNYNTNSSHALRQLRLTQLTEILSGGVYIE(UniProt: P21860-1,KNDKLCHMDTDWRDIVRDRDAEIVVKDNGRSCPPCHEVCKGRCWGPGSEDCQTLIKTICAPQCv1 positions 20 toNGHCFGPNPNQCCHDECAGGCSGPQDTDCFACRHFNDSGACVPRCPQPLVYNKLTFQLEPNP 1342)HTKYQYGGVCVASCPHNFVVDQTSCVRACPPDKMEVDKNGLKMCEPCGGLCPKACEGTGSGSRFQTVDSSNIDGFVNCTKILGNLDFLITGLNGDPWHKIPALDPEKLNVFRTVREITGYLNIQSWPPHMHNFSVFSNLTTIGGRSLYNRGFSLLIMKNLNVTSLGFRSLKEISAGRIYISANRQLCYHHSLNWTKVLRGPTEERLDIKHNRPRRDCVAEGKVCDPLCSSGGCWGPGPGQCLSCRNYSRGGVCVTHCNFLNGEPREFAHEAECFSCHPECQPMEGTATCNGSGSDTCAQCAHFRDGPHCVSSCPHGVLGAKGPIYKYPDVQNECRPCHENCTQGCKGPELQDCLGQTLVLIGKTHLTMALTVIAGLVVIFMMLGGTFLYWRGRRIQNKRAMRRYLERGESIEPLDPSEKANKVLARIFKETELRKLKVILGSGVFGTVHKGVWIPEGESIKIPVCIKVIEDKSGRQSFQAVTDHMLAIGSLDHAHIVRLLGLCPGSSLQLVTQYLPLGSLLDHVRQHRGALGPQLLLNWGVQIAKGMYYLEEHGMVHRNLAARNVLLKSPSQVQVADFGVADLLPPDDKQLLYSEAKTPIKWMALESIHFGKYTHQSDVWSYGVTVWELMTFGAEPYAGLRLAEVPDLLEKGERLAQPQICTIDVYMVMVKCWMIDENIRPTFKELANEFTRMARDPPRYLVIKRESGPGIAPGPEPHGLTNKKLEEVELEPELDLDLDLEAEEDNLATTTLGSALSLPVGTLNRPRGSQSLLSPSSGYMPMNQGNLGESCQESAVSGSSERCPRPVSLHPMPRGCLASESSEGHVTGSEAELQEKVSMCPSRSRSRSPRPRGDSAYHSQRHSLLTPVTPLSPPGLEEEDVNGYVMPDTHLKGTPSSREGTLSSVGLSSVLGTEEEDEDEEYEYMNRRRRHSPPHPRPSSLEELGYEYMDVGSDLSASLGSTQSCPLHPVPIMPTAGTTPDEDYEYMNRQRDGGGPGGDYAAMGACPASEQGYEEMRAFQGPGHQAPHVHYARLKTLRSLEATDSAFDNPDYWHSRLFPKANAQRT 7 Mature humanSEVGNSQAVCPGTLNGLSVTGDAENQYQTLYKLYERCEVVMGNLEIVLTGHNADLSFLQWIREVTHER3 isoform 2GYVLVAMNEFSTLPLPNLRVVRGTQVYDGKFAIFVMLNYNTNSSHALRQLRLTQLTGQFPMVPSG(UniProt: P21860-2 LTPQPAQDWYLLDDDPRLLTLSASSKVPVTLAAVpositions 20 to 133) 8 Mature humanSEVGNSQAVCPGTLNGLSVTGDAENQYQTLYKLYERCEVVMGNLEIVLTGHNADLSFLQWIREVTHER3 isoform 2GYVLVAMNEFSTLPLPNLPVVRGTQWDGKFAIFVMLNYNTNSSHALRQLRLTQLTEILSGGVYIE(UniProt: P21860-3KNDKLCHMDTIDWRDIVRDRDAEIVVKDNGRSCPPCHEVCKGRCWGPGSEDCQTLTKTICAPQCpositions 20 to 331)NGHCFGPNPNQCCHDECAGGCSGPQDTDCFACRHFNDSGACVPRCPQPLVYNKLITQLEPNPHTKYQYGGVCVASCPHNFVVDQTSCVRACPPDKMEVDKNGLKMCEPCGGLCPKAF 9 Human HER3SEVGNSQAVCPGTLNGLSVTGDAENQYQTLYKLYERCEVVMGNLEIVLTGHNADLSFLQWIREVTisoform 1GYVLVAMNEFSTLPLPNLRVVRGTQVYDGKFAIFVMLNYNTNSSHALRQLPLTQLTEILSGGVYIEextracellular regionKNDKLCHMDTIDWRDIVRDRDAEIVVKDNGRSCPPCHEVCKGRCWGPGSEDCQTLTKTICAPQC(UniProt: P21360-1,NGHCFGPNPNQCCHDECAGGCSGPQDTDCFACRHFNDSGACVPRCPQPLVYNKLTEQLEPNPv1 positions 20 toHTKYQYGGVCVASCPHNFVVDQTSCVRACPPDKMEVDKNGLKMCEPCGGLCPKACEGTGSGS 643)RFQTVDSSNIDGFVNCTKILGNLDFLITGLNGDPWHKIPALDPEKLAVERTVREITGYLNIQSWPPHMHNFSVFSNLTTIGGRSLYNRGESLLIMKNLNVTSLGFRSLKEISAGRIYISANRQLCYHHSLNWTKVLRGPTEERLDIKHNRPRPDCVAEGKVCDPLCSSGGCWGPGPGQCLSCRNYSRGGVCVTHCNFLNGEPREFAHEAECFSCHPECQPMEGTATCNGSGSDTCAQCAHFRDGPHCVSSCPHGVLGAKGPIYKYPDVQNECRPCHENCTQGCKGPELQDCLGQTLVLIGKTHLT 10 Human HER3MALTVIAGLVVIFMMLGGTFL isoform 1 transmembrane domain (UniProt:P21860-1, v1 positions 644 to 664) 11 Human HER3YWRGRRIQNKRAMRRYLERGESIEPLDPSEKANKVLARIFKETELRKLKVLGSGVFGTVHKGVWIisoform 1PEGESIKIPVCIKVIEDKSGRQSFQAVTDHMLAIGSLDHAHIVRLLGLCPGSSLQLVTQYLPLGSLLDcytoplasmic domainHVRQHRGALGPQLLLNWGVQIAKGMYYLEEHGMVHRNLAARNVLLKSPSQVQVADFGVADLLPP(UniProt: P21360-1,DDKQLLYSEAKTPIKWMALESIHFGKYTHQSDVWSYGVTVWELMTFGAEPYAGLRLAEVPDLLEKv1 positions 665 toGERLAQPQICTIDVYMVMKCWMIDENIRPTFKELANEFTMARDPPRYLVIKRESGPGIAPGPEP 1342)HGLTNKKLEEVELEPELDLDLDLEAEEDNLATTTLGSALSLPVGTLNRPRGSQSLLSPSSGYMPMNQGNLGESCQESAVSGSSERCPRPVSLHPMPRGCLASESSEGHVTGSEAELQEKVSMCRSRSRSRSPRPRGDSAYHSQRHSLLTPVTPLSPPGLEEEDVNGYVMPDTHLKGTPSSREGTLSSVGLSSVLGTEEEDEDEEYEYMNRRRRHSPPHPPRPSSLEELGYEYMDVGSDLSASLGSTQSCPLHPVPIMPTAGTTPDEDYEYMNRQRDGGGPGGDYAAMGACPASEQGYEEMRAFQGPGHQAPHVHYARLKTLRSLEATDSAFDNNPDYWHSRLFPKANAQRT 12 Human HER3YVVRGRRIQNKRAMRRYLERGESIEPLDPSEKANKYLARIFKETE isoform 1 juxtamembranesegment (UniProt: P21360-1, v1 positions 665 to 708) 13 Human HER3LRKLKVLGSGVFGTVHKGVWIPEGESIKIPVCIKVIEDKSGRQSFQAVTDHMLAIGSLDHisoform 1 proteinAHIVRLLGLCPGSSLQLVTQYLPLGSLLDHVRQHRGALGPQLLLNWGVQIAKGMYYLEEHkinase domainGMVHRNLAARNVLLKSPSQVQVADFGVADLLPPDDKQLLYSEAKTPIKWMALESIHFGKY(UniProt: P21860-1,THQSDVWSYGVTVWELMTFGAEPYAGLRLAEVPDLLEKGERLAQPQICTIDVYMVMVKCWv1 positions 709 to MIDENIRPTFKELANEFT 966) 14 Human HER3RMARDPPRYLVIKRESGPGIAPGPEPHGLTNKKLEEVELEPELDLDLDLEAEEDNLATTTLGSALSisoform 1 C terminalLPVGTLNRPRGSQSLLSPSSGYMPMNQGNLGESCQESAVSGSSERCPRPVSLHPMPRGCLASEsegment (UniProt:SSEGHVTGSEAELQEKVSMCRSRSRSRSPRPRGDSAYHSQRHSLLTPVTPLSPPGLEEEDVNGYP21860-1, v1VMPDTHLKGTPSSREGTLSSVGLSSVLGTEEEDEDEEYEYMNRRRRHSPPHPPRPSSLEELGYEpositions 967 toYMDVGSDLSASLGSTQSCPLHPVPIMPTAGTTPDEDYEYMNRQRDGGGPGGDYAAMGACPASE 1342)QGYEEMRAFQGPGHQAPHVHYARLKTLRSLEATDSAFDNPDYWHSRLFPKANAQRT 15 Human HER3SEVGNSQAVCPGTLNGLSVTGDAENQYQTLYKLYERCEVVMGNLEIVLTGHNADLSFLQWIREVTextracellular regionGYVLVAMNEFSTLPLPNLRVVRGTQVYDGKFAIFVMLNYNTNSSHALRQLRLTQLTEILSGGVYIEsubdomain I KNDKLCHMDTIDWRDIVRDRDAEIVVKDNGRSC (UniProt: P21360-1,v1 positions 20 to 183) 16 Human HER3PPCHEVCKGRCWGPGSEDCQTLTKTICAPQCNGHCFGPNPNQCCHDECAGGCSGPQDTDCFAextracellular regionCRHFNDSGACVPRCPQPLVYNKLTFQLEPNPHTKYQYGGVCVASCPHNFVVDQTSCVRACPPDsubdomain II KMEVDKNGLKMCEPCGGLCPK (UniProt: P21860-1,v1 positions 184 to 329) 17 Human HER3ACEGTGSGSRFQTVDSSNIDGFVNCTKILGNLDFLITGLNGDPWHKIPALDPEKLNVFRTVREITGYextracellar regionLNIQSWPPHMHNFSVFSNLTTIGGRSLYNRGFSLLIMKNLNVTSLGFRSLKEISAGRIYISANRQLCsubdomain III YHHSLNWTKVLRGPTEERLDIKHNRPRRDCVA (UniProt: P21860-1,v1 positions 330 to 495) 18 Human HER3EGKVCDPLCSSGGCWGPGPGQCLSCRNYSRGGVCVTHCNFLNGEPREFAHEAECFSCHPECQextracellular regionPMEGTATCNGSGSDTCAQCAHFRDGPHCVSSCPHGVLGAKGPIYKYPDVQNECRPCHENCTQsubdomain IV GCKGPELQDCLGQTLVLIGKTHLT (UniProt: P21360-1,v1 positions 496 to 643) 19 Human HER3 QPLVYNKLTFQLEPNPHextracellular region subdomain II dimerisation loop (UniProt: P21860-1,v1 positions 261 to 278) 20 Rhesus macaqueMGNLEIVLTGHNADLSFLQWIREVTGYVLVAMNEFSTLPLPNLRVVRGTQVYDGKFAIFVMLNYNTHER3 (UniProt:NSSHALRQLRLTQLTEILSGGVYIEKNDKLCHMDTIDWKDIVRDQDAEIVVKDNGRSCPLCHEVCKF7HEH3-1, v2)GRCWGPGPEDCQTLTKTICAPQCNGHCFGPNPNQCCHDECAGGCSGPQDTDCFACRHFNDSGACVPRCPQPLVYNKLTFQLEPNPHTKYQYGGVCVASCPHNFVVDQTSCVRACPPDKMEVDKNGLKMCEPCGGLCPKACEGTGSGSRFQTVDSSNIDGFVNCTKILGNLDFLITGLNGDPWHKIPALDPEKLNVFRTVREITGYLNIQSWPPHMYNFSVFSNLTTIGGRSLYNRGFSLLIMKNLNVTSLGFRSLKEISAGRIYISANRQLCYHHSLNWTKVLRGPTEERLDIKHNRPRRDCVAEGKVCDPLCSSGGCWGPGPGQCLSCRNYSRGGVCVTHCNFLNGEPREFAHEAECFSCHPECQPMEGTATCNGSGSQTCAQCAHFRDGPHCVSSCPHGVLGAKGPIYKYPDVQNECRPCHENCTQGCKGPELQDCLGQTLVLIGKTHLTMALTVIAGLVVIFMMLGGTFLYWRGRRIQNKRAMRRYLERGESIEPLDPSEKANKVLARIFKETELRKLKVLGSGVFGTVHKGVWIPEGESIKIPVCIKIIEDKSGRQSFQAVTDHMLAIGSLDHAHIVRLLGLCPGSSLQLVTQYLPLGSLLDHVRQHRGALGPQLLLNWGVQIAKGMYYLEEHGMVHRNLAARNVLLKSPSQVQVADFGVADLLPPDDKQLLYSEAKTPIKWMALESIHFGKYTHQSDVWSYGVTVWELMTFGAEPYAGLRLAEVPDLLEKGERLAQPQICTIDWMVMVKCWMIDENIRPTFKELANEFTRMARDPPRYLVIKRESGPGIAPGPEPHGLINKKLEEVELEPELDLDLDLEAEEDNLATTTLGSALSLPVGTLNRPRGSQSLLSPSSGYMPMNQGNLGEAFQESAVSGSSEWCPRPVSLHPMPRGCLASESSEGHVTGSEAELQEKVSTCRSRSRSRSPRPRGDSAYHSQRHSLLTPVTPLSPPGLEEEDVNGYVMPDTHLKGTPSSREGTLSSVGLSSVLGTEEEDEDEEYEYMNRRRRHSPPRPPRPSSLEELGYEYMDVGSDLSASLGSTQSCPLHPVPVMPTAGTTPDEDYEYMNRQRGGSGPGGDYAAMGACPASEQGYEEMRAFQGPGHQAPHVHYAHLKTLRSLEATDSAFDNPDYWHSRLFPKANAQRT 21Epitope recognised YNKLTFQLEPNPH by anti-HER3 antibody done 10A6 22Epitope recognised PRCPQPLVYNKLIT by anti-HER3 antibody clone 4-35-B2 and 4-35-64 23 Composite sequence PRCPQPLVYNKLTFQLEPNPHepitopes recognised by anti-HER3 antibody clones  4-35-62, 4-35-64 and 10A6 24 10D1 heavy chainDVOLQESGPDLVKPSQSLSLTCTVTGYSITSGYSWHWIRQFPGNKLEWMGSIHYSGGTNYNPSLvariable region KSRISITRDTSKNQFFLQLNSVTTEDTATYFCARMTTAPRYPFDYWGQGTTLTVSS25 10D1_c75 heavyDVQLQEWGAGLIKPSETLSLTCAVTGYSITSGYSWHWIRQFPGNGLEWIGSIHYSGGTNYNPTLKchain variable SRITISRDTSKNQFSLKLSSVTAADTAVYFCARMTTAPRYPFDYWGQGTLVTVSSregion 26 10D1_c76 heavyDVQLQEWGAGLLYPSETLSLTCAVTGYSITSGYSWHWIRQFPGNGLEWIGSIHYSGGTNYNPSLKchain variable SRITISRDTSKNQFSLKLSSVTAADTAVYFCARMTTAPRYPFDYWGQGTLVTVSSregion 27 10D1_c77 heavyDVQLQEWGAGLLKPSETLSLTCAVTGYSITSGYSWHWIRQFPGNGLEWIGSIHYSGGTNYNPSLKchain variable SRITISRDTSKNQFSLKLSSVTAADTAVYFCARMTTAPRYPFDYWGOGTLVTVSSregion 28 10D1_c78v1 heavyDVQLQEWGAGLLKPSETLSLTCAVTGYSITSGYSWHWIRQFPGNGLEWIGSIHYSGGTNYNPSLKchain variable SRITISRDTSKNQFSLKLSSVTAADTAVYFCARMTTAPRYPFDYWGOGTLVTVSSregion 29 10D1_c76v2 heavyDVQLQEWGAGLLKPSETLSLTCAVTGYSITSGYSWHWIRQFPGkGLEWIGSIHYSGGTNYNPSLKchain variable SRITISRDTSKNQFSLKLSSVTAADTAVYFCARMTTAPRYPFDYWGQGTLVTVSSregion 30 10D1_11B heavyDVQLQEWGAGLLKPSETLSLTCAVTGYSITSGYSWHWIRQPPGKGLEWIGSIHYSGGTNYNPSLKchain variable SRVTISRDTSKNQFSLKLSSVTAADTAVYYCARMTTAPRYPFDYWGQGTLVTVSSregion 31 10D1_c85v1 heavyDVQLQEWGAGLLKPSETLSLTCAVTGYSITSGYSWHWIRQFPGNGLEWIGSIRYSGGTNYNPSLKchain variable SRITISRDTSKNQFSLKLGSVTAADTAVYFCARMTTAPRYPFDYWGQGTLVTVSSlegion 32 10D1_c85v2 heavyDVQLQEWGAGLLKPSETLSLTCAVTGYSITSGYSWHWIRQFPGKGLEWIGSIRYSGGTNYNPSLKchain variable SRITISRDTSKNQFSLKLGSVTAADTAVYFCARMTTAPRYPFDYWGQGTLVTVSSregion 33 10D1_c85o1 heavyDVQLQEWGAGLLKPSETLSLTCAVTGYSITSGYSWHWIRQFPGKGLEWIGSIRYSGGTNYNPSLKchain variable SRITISRDTSKNQFSLKLGSVTAADTAVYFCARETTAPRYPEDYWGQGTLVTVSSregion 34 10D1_c85o2 heavyDVQLQEWGAGLLKPSETLSLTCAVTGYSITSGYSWHWIRQFPGKGLEWIGSIRYSGGTNYNPSLKchain variable SRITISRDTSKNQFSLKLGSVTAADTAVYFCARGTTAPRYPEDYWGQGTLVTVSSregion 35 10D1_c87 heavyDVOLQEWGAGLLKPSETLSLTCAVTGYSITSGYSWHWIRQFPGNGLEWIGSIHYSGGTNYNPSLKchain variable SRITISRDTSKNQFSLRLSSVTAADTAVYFCARMTTAPRYPFDYWGQGILVIVSSregion 36 10D1_c89 heavyQVQLQESGPGLVKPSQTLSLTCTVSGYSITSGYSWHWIRQHPGKGLEWIGSIRYSGGTDYNPSLKchain variable SLVTISADTSKNQESLKLSSVTAADTAVYYCARMTTAPWYPFDYWGQGTTVTVSSregion 37 10D1_c90 heavyQVQLQESGPGLVKPSQTLFLTCTVSGYSITSGYSWHWIRQHPGKGLEWIGSIRYSGGTDYNPSLKchain variable SLVTISVDTSKNQFSLKLSSVTAADTAVYYCARMTTARNYPFDYWGQGTTVRTSSregion 38 10D1_c91 heavyQVQLQESGPGLVKPSQTLSLTCTVSGYYITSGYSWHWIRQHPGKGLEWIGSIRYSGGTDYNPSLKchain variable SLATISADTSKNQFSLKLSSVTAADTAVYYCARMTTAPWYPFDYWGQGTAVTVSSregion 39 10D1_c92 heavyDVQLQEWGAGLLKPSETLSLTCTVSGYSITSGYSWHWIRQFPGNGLEWIGSIHYSGGTNYNPTLKchain variable SRITISRDTSKNQFSLKLBSVTAADTAVYFCARMTTAPRYPFDYWGQGTLVTVSSregion 40 10D1_c93 heavyDVQLQEWGAGLLKPSETLSLTCAVTGYSITSGYSWHWIRQFPGNGLEWIGSIHYSGGTNYNPSLKchain variable SRITISRDTSKNQFSLRLSSVTAADTAVYFCARMTTAPRYPFDYWGQGTLVTVSSregion 41 10D1, GYSITSGYS 10D1_c75, 10D1_c76, 10D1_c77, 10D1_c78v1,10D1_c78v2, 10D1_11B, 10D1_c85v1, 10D1_c85v2, 10D1_c85o1 , 10D1_c85o2,10D1_c87, 10D1_c89, 10D1_c90, 10D1_c92, 10D1_c93  heavy chain CDR1 4210D1_c91 heavy GYYITSGYS chain CDR1 43 10D1 derived GYX₁ITSGYSconsensus heavy wherein X₁ = S or Y chain CDR1 44 10D1, IHYSGGT10D1_c75, 10D1_c76, 10D1_c77, 10D1_c78v1, 10D1_c78v2, 10D1_11B,10D1_c87, 10D1_c92, 10D1_c93 heavy chain CDR2 45 10D1_c85v1, IRYSGGT10D1_c85v2, 10D1_c85o1, 10D1_c85o2, 10D1_c99, 10D1_c90, 10D1_c91 heavychain CDR2 46 10D1 derived IX₂YSGGT consensus heavy wherein X₂ = H or Rchain CDR2 47 10D1, ARMTTAPRYPFDY 10D1_c75, 10D1_c76, 10D1_c77,10D1_c78v1, 10D1_c78v2, 10D1_11B, 10D1_c85v1, 10D1_c85v2, 10D1_c87,10D1_c92, 10D1_c93 heavy chain CDR3 48 10D1_c89, ARMTTAPWYPFDY 10D1_c90,10D1_c91 heavy chain CDR3 49 10D1_c85o1 ARETTAPRYPFDY chain CDR3 5010D1_c85o2 heavy ARGTTAPRYPFDY chain CDR3 51 10D1 derivedARX₃TTAPX₄YPFDY consensus heavy wherein X₃ = M, E or G; X₄ = R or Wchain CDR3 52 10D1_c75 DVQLQEWGAGLLKPSETLSLTCAVT 10D1_c76, 10D1_c77,10D1_c78v1, 10D1_c78v2, 10D1_c85v1, 10D1_c85v2, 10D1_c85o1, 10D1_c85o2,10D1_c87, 10D1_c92, 10D1_c93 heavy chain FR1 53 10D1_c89,QVQLQESGPGLVKPSQTLSLTCTVS 10D1_c91 heavy chain FR1 54 10D1_c90 heavyQVQLQESGPGLVKPSQTLFLTCTVS chain FR1 55 10D1 heavy chainDVQLQESGPDLVKPSQSLSLTCTVT FR1 56 10D1_c75 WHWIRQFPGNGLEWIGS 10D1_c76,10D1_c77, 10D1_c78v1, 10al_c85v1, 10D1_c87, 10D1_c92, 10D1_c93 heavychain FR2 57 10D1_c78v2, WHWIRQFPGKGLEWIGS 10D1_c85v2, 10D1_c85o1,10D1_c85o2 heavy chain FR2 58 10D1 heavy chain WHWIRQFPGNKLEWMGS FR2 5910D1_c89, WHWIROARGKGLEWIGS 10D1_c90, 10D1_c91 heavy chain FR2 6010D1_11B heavy WHWIRQFPGKGLEWIGS chain FR2 61 10D1_c75,NYNPTLKSRITISRDTSKNQFSLKLSSVTAADTAVYFC 10D1_c92 heavy chain FR3 6210D1_c76, NYNPSLKSRITISRDTSKNOFSLKLSSVTAADTAVYFC 10D1_c77, 10D1_c78v1,10D1_c78v2 heavy chain FR3 63 10D1_11B heavyNYNPSLKSRVTISRDTSKNQFSLKLSSVTAADTAVYYC chain FR3 64 10D1_085v1,NYNPSLKSRITISRDTSKNQFSLKLGSVTAADTAVYFC 10D1_c85v2, 10D1_c8501,10D1_c8502 heavy chain FR3 65 10D1_c87,NYNPSLKSRITISRDTSKNQFSLRLSSVTAADTAVYFC 10D1_c93 chain FR3 6610D1_c89 heavy DYNPSLKSLVTISADTSKNQFSLKLSSVFAADTAVYYC chain FR3 6710D1_c90 heavy DYNPSLKSLVTISVDTSKNQFSLKLSSVTAADTAVYYC chain FR3 6810D1_c91 heavy DYNPSLKSLATISADTSKNQFSLKLSSVTAADTAVYYC chain FR3 6910a1 heavy chain NYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYFC FR3 70 10D1_c75,WGQGTLVTVSS 10D1_c76, 10D1_c77, 10D1_c78v1, 10D1_c78v2, 10D1_11B,10D1_c85v1, 10D1_c85v2, 10D1_c85o1, 10D1_c85o2, 10D1_c87, 10D1_c92,10D1_c93 heavy chain FR4 71 10D1_c89, WGQGTTLTVSS 10D1_c90 heavychain FR4 72 10D1_c91 heavy WGQGTAVTVSS chain FR4 73 10D1, 4-35-F34,WGQGTTLTVSS 10A6 heavy chain FR4 74 10D1 light chainDIVMTQSQKFMSTSVGDRVSVTCKASQIVGSNVAWYQQKPGQSPKPLIYSASYRYSGVPDRFTAvariable region SGSGTDFTLTITNVQSEDLAEYFCQQYSSHPLTFGAGTKLELK 7510D1_c75 lightDIVMTQSPSSLSASVGDLVTITCKASQIVGSNVAWYQMKPGKSPKPLIYSASYLYFGVPSRFSGSGchain variable SGTDFTLTISSLQPEDVAEYFCQQYSSHPLTFGPGIKVEIK region 7610D1_c78 lightDIVMTQSPSSLSASGGDRVTITCKASQIVGYNVAWYQQKPGKSPKPLIYSASYLYSDVPSRFSASchain variable GSGTDFTLTISSLQPEDVAEYFCQQYSSHPLTFGPGTKVEIK region 7710D1_c77 lightVIVMTQSPSSLSASVGDRVTITCKASQIVGPNVAWYQQKPGKSPKPLIYSASYGYSDVPSRFSGSchain variable GSGTDFTLTISSLQPEDVAEYFCQQYSTHPLTFGPGTKVEIK region 7810D1_c78v1,DIVMTQSPSSLSASVGDRVTITCKASQIVGSNVAWYQQKPGKSPKPLIYSASYGYSDVPSRFSGS10D1_c78v2, GSGTDFTLTISSLRPEDVATYYCQQYSSHPLTFGRGTKVEIK 10D1_11B lightchain variable region 79 10D1_c85v1,DIVMTQSPSSLSASVGDRVTITCKASQIVGENVAWYQQKPGKSPKPLIYSARYQYSGVPFRFSGS10D1_c85v2 light GSGTDFTLTISSLQPEDVATYYCQQYSSHPLTFGPGTKVEIKchain variable region 80 10D1_c35o1 lightDIVMTQSPSSLSASVGDRVTITCKASQIVGSNVAWYQQKPGKSPKPLIYSARYQYSGVPFRFSGSchain variable GSGTDFTLTISSLQPEDVATYYCQQYSSHPLTFGPGTKVEIK region 8110D1_c85o2 lightDIVMTQSPSSLSASVGDRVTITCKASQIVGENVAWYQQKPGKSPKPLIYSARYQYSGVPFRFSGSchain variable GSGTDFTLTISSLQPEDVATYYCQQYSSHPLTFGPGTKVEIK region 8210D1_c37 lightDIVMTQSPSSLSASVGDRVTITCKASQIVGSNVAWYQQMPGKSPEPLIYSASYLYSDVPSRFSGSchain variable GSGTDFTMTISSLQPEDVATYYCQQYSSHPLTFGPGTKVEIK region 8310D1_c89 lightDIQMTQSPSSVSASVGDRVTITCKASQIVGSNVAWYQQKPGKAPEPLIYSASYLYSGVPSRFSGSchain variable GSGTDFTLTISSLQPEDFATYYCQQYSSHPLIFGQGTKLEIK region 8410D1_c90 lightDIQMTQSPSSVSASVGDRVTITCKASQIVGSNVAWYQQKPGKAPEPLIYSASYLYSSVPSRFSGSchain variable GSGTEFTMTISSLEPEDFATYYCQQYTTHPLTFGRGTKVEIK region 8510D1_c91 lightDIQMTQSPSSVSASVGDRVTITCKASQIVGSNVAWYQQKPGKAPMPLIYSASYGYSGVPSRFSGSchain variable GSGTDFTLTISSLQPEDFATYYCQQYSSIIPLTFGQGTKLEIK region 8610D1_c92 lightDIVMTQSPSSVSASVGDLVTITCKASQIVGSNVAWYQMKLGKSPKPLIYSASYLYFGVPSRFSGSGchain variable SGTDFTLITSSLQPEDVAEYFCQQYFSHPLTFGPGTKVEIK region 8710D1_c93 lightDIVMTQSPSSLSASVGDRVTITCKASQIVGSNVAWYQQKPGKSPKPLIYSASYLYSDVPSRFSGSchain Variable GSGTDFTMTISSMPEDVATYYCQQYSSHPLTFGRGTKVEIK region 88 10D1,QIVGSN 10D1_c75, 10D1_c78v1, 10D1_c78v2, 10D1_11B, 10D1_c35v1,10D1_c65v2, 10D1_c65ol, 10D1_c85o2, 10D1_c87, 10D1_c89, 10D1_c90,10D1_c91, 10D1_c92, 10D1_c93 light chain CDR1 89 10D1_c76 light QIVGYNchain CDR1 90 10D1_c77 light QIVGPN chain CDR1 91 10D1 derived QIVGX₅Nconsensus light wherein X₅ = S, Y or P chain CDR1 92 10D1, SAS c75,10D1_c76, 10D1_c77, 10D1_c78v1, 10D1_c78v2, 10D1_11B, 10D1_c87,10D1_c89, 10D1_c90, 10D1_c91, 10D1_c92, 10D1_c93 light chain CDR2 9310D1_c85v1, SAR 10D1_c85v2, 10D1_c85o1, 10D1_c85o2 light chain CDR2 9410D1 derived SAX₆ consensus light wherein X₆ = S or R chain CDR2 9510D1, QQYSSHRLT 10D1_c75, 10D1_c76, 10D1_c78v1, 10D1_c73v2, 10D1_116,10D1_c85v1, 10D1_c85v2, 10D1_c8501, 10D1_c85o2, 10D1_c87, 10D1_c39,10D1_c91, 10D1_c93 light chain CDR3 96 10D1_c77 light QQYSTHPLTchain CDR3 97 10D1_c90 light QQYTTHPLT chain CDR3 98 10D1_c92 lightQQYFSHPLT chain CDR3 99 10D1 derived QQYX₇X₈HPLT consensus lightwherein X₇ = S, T or F; X₈ = S or T chain CDR3 100 10D1_c75,DIVMTQSPSSLSASVGDLVTITCKAS 10D1_c92 light chain FR1 101 10D1_c76 lightDIVMTQSPSSLSASGGDRVTITCKAS chain FR1 102 10D1_c77 lightVIVMTQSRSSLSASVGDRVTITCKAS chain FR1 103 10D1_c78v1,DIVMTQSPSSLSASVGDRVTITCKAS 10D1_c78v2, 10D1_11B, 10D1_c85v1, 10D1_c85v2,10D1_c85o1, 10D1_c85o2, 10D1_c87, 10D1_c93 light chain FR1 104 10D1_c89,DIQMTQSPSSVSASVGDRVTITCKAS 10D1_c91 chain FR1 105 10D1_c90 lightDIQMTQSPSSVSASVGDRVTFTCKAS chain FR1 106 10D1 light chainDIVMTQSQKFMSTSVGDRVSVTCKAS FR1 107 10D1_c75 light VAWYQMKPGKSPKPLIYchain FR2 108 10D1_c76, VAWYQQKPGKSPKPLIY 10D1_c77, 10D1_c78v1,10D1_c78v2, 10D1_11B, 10D1_c85v1, 10D1_c85v2, 10D1_c85o1 , 10D1_c85o2,10D1_c93 light chain FR2 109 10D1_c87 light VAWYQQMPGKSPEPLIY chain FR2110 10D1_c89, VAWYQQKPGKAPEPLIY 10D1_c90 light chain FR2 11110D1_c91 light VAWYQQKPGKAPMPLIY chain FR2 112 10D1_c92 lightVAWYQMKLGKSPKPLIY chain FR2 113 10D1 light chain VAWYQQKPGQSPKPLIY FR2114 10D1_c75, YLYFGVPSRFSGSGSGTDFTLTISSLQPEDVAEYFC 10D1_c92 lightchain FRS 115 10D1_c76 light YLYSDVPSRFSASGSGTDFTLTISSLQPEDVAEYFCchain FR3 116 10D1_c77 light YGYSDVPSRFSGSGSGTDFTLTISSLQPEDVAEYFCchain FR3 117 10D1_c78v1 YGYSDVPSRFSGSGSGTDFTLTISSLRPEDVATYYC10D1_c78v2, 10D1_11B light chain FR3 118 10D1_c85v1,YQYSGVPFRFSGSGSGTDFTLTISSLQPEDVATYYC 10D1_c85v2, 10D1_c85o1,10D1_c85o2 light chain FR3 119 10D1_c87,YLYSDVPSRFSGSGSGTDFTMTISSLQPEDVATYYC 10D1_c93 light chain FR3 12010D1_c89 light YLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYD chain FR3 12110D1_c90 light YLYSSVPSRFSGSGSGTEFTMTISSLEPEDFATYYC chain FR3 12210D1_c91 light YGYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC chain FR3 12310D1 light chain YRYSGVPDRFTASGSGIDFTLATINVOSEDLAEYFC FR3 124 10D1_c75,FGPGTKVEIK 10D1_c76, 10D1_c77, 10D1_c78v1, 10D1_c78v2, 10D1_11B,10D1_c85v1, 10D1_c65v2, 10a1_c65o1, 10D1_c8502, 10D1_c87, 10D1_c90,10D1_c92, 10D1_c93 light chain FR4 125 10D1_c69, FGQGTKLEIK10D1_c91 light chain FR4 126 10D1 light chain FGAGTKLELK FR4 1274-35-B2 heavyEIQLQQSGPELVKPGASVKVSCKASGYSFTDYNMYWVKQSHGKSLEWIGHINPYNGGTTYNQKFchain variable KGRATLTVDKSSSTAFMHLNSLTSEDSAVYFCVSLRWGAMDYWGQGTSVTVSSregion 128 4-35-B2 heavy GYSFTDYN chain CDR1 129 4-35-B2 heavy INRYNGGTchain CDR2 130 4-35-B2 heavy VSLRWGAMDY chain CDR3 131 4-35-B2 heavyEIQLQQSGPELVKPGASVKVSCKAS chain FR1 132 4-35-B2 heavy MYWVKQSHGKSLEWIGHchain FR2 133 4-35-B2 heavy TYNQKFKGRATLTVDKSSSTAFMHLNSLTSEDSAVYFCchain FR3 134 4-35-B2 heavy WGQGTSVTVSS chain FR4 1354-35-B2 light chainQIVLTOSPALMSASPGEKVTMTCSASSSVSYMYWYQQKPRSSPKPWIYLTSNLASGVPARFSGSvariable region GSGTSYSLTISSMEAEDAATYYCQQWNSNPYTFGGGTKLEIK 1364-35-B2 light chain SSVSY CDR1 137 4-35-B2 light chain LTS CDR2 1384-35-B2 light chain QQWNSNPYT CDR3 139 4-35-B2 light chainQIVLTQSPALMSASPGEKVTMTCSAS FR1 140 4-35-B2 light chain MYWYQQKPRSSPKPWIYFR2 141 4-35-B2 light chain NLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYC FR3 1424-35-B2, 4-35-B4, FGGGTKLEK 10A6 light chain FR4 143 4-35-B4 heavyEVQLQQSGAELVKPGASVKLSCTASGNIKDTYIHWVKQRPDQGLEWIGKIIDPANGNTNYDPKFchain variable QGKATITADTSSNTAYLQLSSLSSEDTAWFCARGLHWGQGTTLTVSS region144 4-35-B4 heavy GFNIKDTY chain CDR1 145 4-35-B4 heavy IDPANGNTchain CDR2 146 4-35-B4 heavy ARGLH chain CDR3 147 4-35-B4 heavyEVQLQQSGAELVKPGASVKLSCTAS chain FR1 148 4-35-B4 heavy IHWVKQRPDQGLEWIGKchain FR2 149 4-35-B4 heavy NYDPKFQGKATITADTSSNTAYLQLSSLSSEDTAVYFCchain FR3 150 4-35-B4 light chainDIVLTQSPASLAVSLGQRATISCRASKSVSTSGYSYMHWYQQKPGQPPKLLYLASNLESGVPARFvariable region SGSGSGTDFTLNIHRVEEEDAATYYCQHSRELPYTFGGGTKLEIK 1514-35-B4 light chain KSVSTSGYSY CDR1 152 4-35-B4 light chain LAS CDR2 1534-35-B4 light chain QHSRELPYT CDR3 154 4-35-B4 light chanDIVLTQSPASLAVSLGQRATISCRAS FR1 155 4-35-B4 light chain MHWYQQKPGQPPKLLIYFR2 156 4-35-B4 light chain NLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYC FR3 1574-35-B4 light chainDVQLQESGPGLVKPSQSLSLTCSVTGNFITSGYFWNWIRQFPGNKLEWMGHSYDGSNNYKPSLvariable region KNRISITRDTSKNQFFLKLNSVTTEDTATYYCARENYGFGFDYWGQGTTLTVSS158 10A6 heavy chain GNFITSGYF CDR1 159 10A6 heavy chain ISYDGSN CDR2160 10A6 heavy chain ARENYGFGFDY CDR3 161 10A6 heavy chainDVQLQESGPGLVKPSQSLSLTCSVT FR1 162 10A6 heavy chain WNWIRQFPGNKLEWMGF FR2163 10A6 heavy chain NYKPSLKNRISITRDTSKNQFFLKLNSVTTEDTATYYC FR3 16410A6 light chainDIVLTQSPSSLPVSIGEKVTMSCKSSQSLLYSDNQKNYLAWYQQKPGQSPKLLIYWASTWKSGVPvariable region DRFTGSGSGIDFTLTISSVKAEDLAVYYCQQYFTFPWIFGGGTKLEIK 16510A6 light chain QSLLYSDNQKNY CDR1 166 10A6 light chain WAS CDR2 16710A6 light chain QQYFTFPWT CDR3 168 10A6 light chainDIVLTQSPSSLPVSIGEKVTMSCKSS FR1 169 10A6 light chain LAVWQQKPGQSPKLLIYFR2 170 10A6 light chain TWKSGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYC FR3 171Human IgG1ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLconstant regionSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPFKP(IGHG1; UniProt:KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQP01857-1, v1)DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGDPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK172 CH1 IgG1 (positionsASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFFAVLQSSGLYSL1-98 of P01857-1, SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV v1) 173 Hinge IgG1EPKSCDKTHTCP (positions 99-110 of P01857-1, v1) 174 HC2 IgG1 (positionsPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPR111-223 of  EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKP01357-1, v1) 175 CH3 IgG1 (positionsGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF224-330 of  FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK P01857-1, v1)176 CH3 (D3563,GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFL358M; positions FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK numberedaccording to EU numbering) 177 Cκ CL (IGCK:RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSUniProt:  TYSLSSTLTLSKADYEKHKWACEVTHQGLSSPVTKSFNRGEC P01834-1, v2) 17810A6 heavy chain MKVLSLLYLLTAIPGILS signalP 179 10D1 heavy chanMRVLILLCLFTAFPGILS SignalP 180 10D1 light chain MESQTQVFVYMLLWLSGVDGSignalP 181 4-35-B2 heavy MEWSWIFLFLLSGTTGVHS chain SignalP 1824-35-B2 light chain MDFQVQIF6FLLMSASVMMSRG SignalP 183 4-35-B4 heavyMKCSWVIFFLMAVVTGVNB chain SignalP 184 4-35-B4 light chainMETDTLLLWVLLLWVPGSTG SignalP 185 10D1_c75, MELGLRVWFLIATLAGARC 10D1_c76,10D1_c77, 10D1_c78v1, 10D1_c78v2, 10D1_11B, 10D1_c85v1, 10D1_c85v2,10D1_c8501 , 10D1_c85o2, 10D1_c87, 10D1_c89, 10D1_c90, 10D1_c91 ,10D1_c92, 10D1_c93 heavy chain SignalP 186 10D1_c75,MDMRVPAQLLGLLLLWLRGARC 10D1_c76, 10D1_c77, 10D1_c76v1, 10D1_c78v2,10D1_11B, 10D1_c85v1, 10D1_c85v2, 10D1_c85o1, 10D1_c85o2, 10D1_c67,10D1_c69, 10D1_c90, 10D1_c91, 10D1_c92, 10D1_c93 light chain SignalP 18710D1_c75 VH-CH1-DVQLQEWGAGLLKPSETLSLTCAVTGYSITSGYSWHWIRQFPGNGLEWIGSIHYSGGTNYNPTLKCH2-CH3SRITISRDTSKNQFSLKLSSVTAADTAVYFCARMTTAPRYPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFREPVTVSWNSGALTSGVHTFPAVLQBSGLYSLBSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 18810D1_c75 VL-CκDIVMTQSPSSLSASVGDLVTITCKASQIVGSNVAWYQMKPGKSPKPLIYSASYLYFGVPSRFSGSGSGTDFTLTISSLQPEDVAEYFCQQYSSHPLTFGPGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 189 10D1_c76 VH1-CH1-DVQLQEWGAGLLKPSETLSLTCAVTGYSITSGYSWHWIRQFPGNGLEWIGSHYSGGTNYNPSLK CH2-CH3SRIISRDTSKNQFSLKLSSVTAADTAVYFCARMTTAPRYPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKERWQQGNVFSCSVMHEALHNHYTQKSLBISPGK 19010D1_c76 VL-CκDIVMTQSPSSLSASGGDRVTITCKASQIVGYNVAMOQKPGKSPKPLIYBASYLYBDVPSRFSASGSGTDFTLTISSLQPEDVAEYFCQQYSSHPLITGPGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTISKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 191 10D1_c77 VH1-CH1-DVQLQEWGAGLLKPBETLSLTCAVTGYSITSGYSWHWIRQFPGNGLEWIGSIHYSGGTNYNPSLKCH2-CH3SRITISRDTSKNQFSLKLSSVTAADTAVYFCARMTTAPRYPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 19210Da_c77 VL-CκVIVMTQSPSSLSASVGDRVTITCKASQIVGPNVAWYQQKPGKSPKPLIYSASYGYSDVPSRFSGSGSGTDFTLTISSLQPEDVAEYFCQQYSTHPLTFGPGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSILTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 193 10D1_c78v1 VH-DVQLQEWGAGLLKPSETLSLTCAVTGYSITSGYSWHWIRQFPGNGLEWIGSIHYSGGTNYNPSLKCH1-CH2-CH3SRITISRDTSKNQFSLKLSSVTAADTAVYFCARMTTAPRYPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKEKVSNKALPAPIEKTISKAKGQPREPQWTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 19410D1_c78v2 VH-DVQLQEWGAGLLKPSETLSLTCAVTGYSITSGYSWHWIRQFPGkGLEWIGSIHYSGGTNYNPSLKCH1-CH2-CH3SRITISRDTSKNQFSLKLSSVTAADTAVYFCARMTTAPRYPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKERWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 19510D1_c78v1,DIVMTQSPSSLSASVGDRVTITCKASQIVGSNVAWYQQKPGKSPKPLIYSASYGYSDVPSRFSGS10D1_c78v2,GSGTDFTLTISSLRPEDVATYYCQQYSSHPLTFGPGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASV10D1_11B VL-CκVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 196 10D1_11B VH-DVQLQEWGAGLLKPSETLSLTCAVYGYSITSGYSWHWIRQFPGKGLEWIGSHYSGGTNYNPSLKCH1-CH2-CH3SRVTISRDTSKNQFSLKLSSVTAADTAVYYCARMTTAPRYPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQWTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 19710D1_c85v1 VH-DVQLQEWGAGLLKPSETLSLTCAVTGYSITSGYSWHWIRQFPGNGLEWIGSRYSGGTNYNPSLKCH1-CH2-CH3SRITISRDTSKNQFSLKLGSVTAADTAVYFCARMTTAPRYPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 19810D1_c85v2 VH-DVQLQEWGAGLLKPSETLSLTCAVTGYSITSGYSWHWRQFPGKGLEWIGSIRYSGGTNYNPSLKCH1-CH2-CH3SRITISRDTSKNQFSLKLGSVTAADTAVYFCARMTTAPRYPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVFPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 19910D1_c85v1,DIVMTQSPSSLSASVGDRVTEICKASQIVGSNVAWYQQKPGKSPKPLIYSARYQYSGVPFRFSGS10D1_c85v2 VL-CκGSGTDFTLTISSLQPEDVATYYCQQYSSHPLTFGPGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 200 10D1_c8501 VH-DVQLQEWGAGLLKPSETLSLTCAVTGYSITSGYSWHWIRQFPGKGLEWIGSIRYSGGTNYNPSLKCH1-CH2-CH3SRITISFRDTSKNQFSLKLGSVTAADTAVYFCARETTAPRYPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 20110D1 _c85o1 DIVMTQSPSSLSASVGDRVTITCKASQIVGSNVAWYQQKPGKSPKPLIYSARYQYSGVPFRFSGS VL-CκGSGTDFTLTISSLQPEDVATYYCQQYSSHPLTFGPGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 202 10D1_c85o2DVQLQEWGAGLLKPSETLSLCAVTGYSITSGYSWHWIRQFPGKGLEWIGSIRYSGGTNYNPSLKVH-CH1-CH2-CH3SRITISRDTSKNQFSLKLGSVTAADTAVYFCARGTTAPRYPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVOKKVEPKSCDKTHICPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKEKVSNKALPAREKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGEYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFELYSKLTVDKSRWQQGNVESCSVMHEALHNHYTQKSISLSPGK 20310D1 _c85o2 DIVMTQSPSSLSASVGDRVTITCKASOIVGSNVAVVYQQKPGKSPKPLEYSARYQYSGVPFRFSGS VL-CκGSGTDFTLTISSLQPEDVATYYCQQYSSHPLTFGPGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSPNRGEC 204 10D1_c87 DVQLQEWGAGLLKPSETLSLTCAVTGYSITSGYSWHWIRQFPGNGLEWIGSIHYSGGTNYNPSLKVH-CH1-CH2-CH3SRITISRDTSKNQFSLRLSSVTAADTAVYPCARMTTAPRYPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 20510D1_c87 VL-CκDIVMTOSPSSLSASVGDRVTITCKASQIVGSNVAWYQQMPGKSPEPLIYSASYLYSDVPSRFSGSGSGTDFTMTISLQPEDVATYYCQQYSSHPLTFGPGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 206 10D1_c89 QVQLQESGPGLVKPSQTLSLTCTVSGYSITSGYSWHWIRQHPGKGLEWIGSIRYSGGTDYNPSLKVH-CH1-CH2-CH3SLVTISADTSKNQFSLKLSSVTAADTAVYYCARMTTAPWYPFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLEPPKPKDTLMSRTPEVTCVVVDVSHEDPEVKFNWWDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSPFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 20710D1_c89 VL-CκDIQMTQSPSSVSASVGDRVTITCKASQIGSNVAWYQQKPGKAPEPLYSASYLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSSHPLTPGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSILTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 205 10D1_c90 QVQLQESGPGLVKPSQTLFLTCTVSGYSITSGYSWHWIRQHPGKGLEWIGSIRYSGGTDYNPSLKVH-CH1-CH2-CH3SLVTISVDTSKNQFSLKLSSVTAADTAVYYCARMTTAPWYPFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGQTYICNVNHKPSNTKVDKKVEPKSCDKTHTEPPCPAPELLGGPSVFLPPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 20910D1_c90 VL-CκDIQMTQSPSSVSASVGDRVTFTCKASQIVGSNVAWYQQKPGKAPEPLIYSASYLYSSVPSRFSGSGSGTEFTMTISSLEPEDFATYYCQQYTTHPLTFGPGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 210 10D1_c91 QVQLQESGPGLVKPSQTLSLTCTVSGYYITSGYSWHWIRQHPGKGLEWIGSIRYSGGTDYNPSLKVH-CH1-CH2-CH3SLATISADTSKNQFSLKLSSVTAADTAVYYCARMTTAPWYPFDYWGQGTAVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGLTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGPYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFELYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTOKSLSLSPGK 21110D1_c91 VL-CκDIQMTQSPSSVSASVGDRVTITCKASQIVGSNVAWYQQKPGKAPMPLIYSASYGYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSSHPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSPNRGEC 212 10D1_c92 DVQLQEWGAGLLKPSETLSLTCAVTGYSITSGYSWHWRQFPGNGLEWIGSIHYSGGTNYNPTLKVH-CH1-CH2-CH3SRITISRDTSKNQFSLKLSSVTAADTAVYFCARMTTAPRYPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVPLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 21310D1_c92 VL-CκDIVMTQSPSSLSASVGDLVTITCKASQNGSNVAWYQMKLGKSPKPLIYSASYLYFGVPSRFSGSGSGTDFTLTISSLQPEDVAEYFCQQYFSHPLTFGPGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 214 10D1_c93 DVQLQEWGAGLLKPSETLSLTCAVTGYSITSGYSWHWIRQFPGNGLEWIGSHYSGGTNYNPSLKVH-CH1-CH2-EH3SRITISRDTSKNQFSLRLSSVTAADTAWFCARMTTAPRYPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKIHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRIPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHODWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTIPPSRDELIKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSPFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSISPGK 21510D1_c93 VL-CκDIVMTQSPSSLSASVGDRVTITCKASQIVGSNVAWYQQKPGKSPKPLIYSASYLYSDVPSRFSGSGSGTDFTMTISSLQPEDVATYYCQQYSSHPLTFGPGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 216 10D1 VH-CH1-DVQLQESGPDLVKPSQSLSLTCTVTGYSITSGYSWHWIRQFPGNKLEWMGSIHYSGGTNYNPSL CH2-CH3KSRISITRDTSKNQFFLQLNSVTTEDTATYFCARMTTAPRYPFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 21710D1 VL-CκDIVMTQSQKFMSTSVGDRVSVTCKASQIVGSNVAWYQQKPGQSPKPLIYSASYRYSGVPDRFTASGSGTDFTLTITNVQSEDLAEYFCQQYSSHPLTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQCSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSRVTKSFNRGEC 218 4-35-B2EIQLQQSGPELVKPGASVKVSCKASGYSFTDYWVYWVKQSHGKSLEWIGHINPYNGGTTYNQKFVH-CH1-CH2-CH3 KGRATLTVDKSSSTAFMHLNSLTSEDSAVYFCVSLRVVGAMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKRKDTLMISRTREVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 2194-35-B2 VL-CκQIVLTQSPALMSASPGEKVTMTCSASSSVSYMYWYQQKPRSSPKPWIYLTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWNSNPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTA   SVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 220 4-35-B4 VEVQLQQSGAELVKPGASVKLSCTASGFNIKDTYIHWVKQRPDQGLEWIGKIIDPANGNTNYDPKFH-CH1-CH2-CH3QGKATITADTSSNTAYLQLSSLSSEDTAVYFCARGLHVVGQGTTLTVSSASTKGPSVPPLAPSSKSTSGGTAALGCLVKDYFFEPVTVSWNSGALTSGVHTFPAVWSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPFKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 221 4-35-B4 VL-CκDIVLTQSPASLAVSLGQRATISCRASKSVSTSGYSYMHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPPEAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 222 10A6 VH-CH1-DVQLQESGPGLVKPSQSLSLTCSVTGNFITSGYFWNWIRQFPGNKLEWMGFISYDGSNNYKPSL CH2-CH3KNRISITRDTSKNQFFLKLNSVTTEDTATYYCARENYGFGFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPARELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 22310A6 VL-CκDIVLTQSPSSLPVSIGEKVTMSCKSSQSLLYSDNQKNYLAWYQQKPGQSPKLLIYWASTWKSGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYFTFPWTFGGGTKLEIKRTVAARSVFIFFPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 224 10D1_11B heavy DVQLQEWGAGLLKPSETLSLTCAVYchain FR1 225 10D1F VH-CHI-QVQLQESGPGLVKPSQTLSLTCTVSGYSITSGYSWHWIRQHPGKGLWIGSIRYSGGTDYNPSLK CH2-CH3SLVTISADTSKNQRSLKLSSVTAADTAVYYCARMTTARWYPFDYANGQGTIVTVSSASTKGPSVFP(GASDALIE; LCKC)LAPSSKSTSGGTAALGCLVKDYFREPVTVSANNSGALTSGVHTFRAVLQSSGLYSLSSVVTVPSSS(10D1F.FcB)LGTQTYTCNVNHKRSNTKVDKKVEPKSCDKTHTCPPCPAPELLAGPDVFCFPPKRKDTLMISRTREVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPLPEECTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 22610D1F VH-CH1-QVQLQESGPGLVKPSQTLSLTCTVSGYSITSGYSWHIMRQHPGKGLEWIGSIRYSGGTDYNPSLKCH2-CH3 (N297Q)SLVTISADTSKNQFSLKLSSVTAADTAVYYCARMTTAPWYPFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQP  ENNYKTTPRVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTOKSLSLSPGK 22710D1 VH-CH1-DVQLQESGPDLVKPSQSLSLTCTVTGYSITSGYSWHIMRQFPGNKLEWMGSIHYSGGTNYNPSL CH2-CH3KSRISITRDTSKNQFFLQLNSVTTEDTATYFCARMTTAPRYPFDYWGQGTTLTVSSASTKGPSVFP(GASDALIE; LCKC) LAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVERKSCDKTHTCPPCPARELLAGRDVFCFPPKPKDTLMISRIPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPLPEECTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSISLSPGK 22810D1 VH-CH1-DVQLQESGPDLVKPSQSLSLTCTVTGYSITSGYSWHWIRQFPGNKLEMAGSEHYSGGTNYNPSLCH2-CH3 (GASD)KSRISITRDTSKNQFFLQLNSVTTEDTATYFCARMTTAPRYPFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLAGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 229HER3 binding site CFGPNPNQCCHDECAGGC for 10D1-derived clones 230Binding site  PNPNQ motif 1 231 Binding site  DECAG motif 2

Numbered Paragraphs

The following numbered paragraphs (paras) provide further statements offeatures and combinations of features which are contemplated inconnection with the present invention:

1. An antigen-binding molecule, optionally isolated, which is capable ofbinding to HER3 in extracellular region subdomain II.

2. The antigen-binding molecule according to para 1, wherein theantigen-binding molecule inhibits interaction between HER3 and aninteraction partner for HER3.

3. The antigen-binding molecule according to para 1 or para 2, whereinthe antigen-binding molecule is capable of binding to a polypeptidecomprising or consisting of the amino acid sequence of SEQ ID NO:16.

4. The antigen-binding molecule according to any one of paras 1 to 3,wherein the antigen-binding molecule is capable of binding to apolypeptide comprising the amino acid sequence of SEQ ID NO:23 or SEQ IDNO:229.

5. The antigen-binding molecule according to any one of paras 1 to 4,wherein the antigen-binding molecule is capable of binding to apolypeptide comprising the amino acid sequence of SEQ ID NO:21 or SEQ IDNO:229.

6. The antigen-binding molecule according to any one of paras 1 to 5,wherein the antigen-binding molecule comprises:

-   -   (i) a heavy chain variable (VH) region incorporating the        following CDRs:        -   HC-CDR1 having the amino acid sequence of SEQ ID NO:43        -   HC-CDR2 having the amino acid sequence of SEQ ID NO:46        -   HC-CDR3 having the amino acid sequence of SEQ ID NO:51; and    -   (ii) a light chain variable (VL) region incorporating the        following CDRs:        -   LC-CDR1 having the amino acid sequence of SEQ ID NO:91        -   LC-CDR2 having the amino acid sequence of SEQ ID NO:94        -   LC-CDR3 having the amino acid sequence of SEQ ID NO:99.

7. The antigen-binding molecule according to any one of paras 1 to 6,wherein the antigen-binding molecule comprises:

-   -   (i) a heavy chain variable (VH) region incorporating the        following CDRs:        -   HC-CDR1 having the amino acid sequence of SEQ ID NO:41        -   HC-CDR2 having the amino acid sequence of SEQ ID NO:44        -   HC-CDR3 having the amino acid sequence of SEQ ID NO:47; and    -   (ii) a light chain variable (VL) region incorporating the        following CDRs:        -   LC-CDR1 having the amino acid sequence of SEQ ID NO:88        -   LC-CDR2 having the amino acid sequence of SEQ ID NO:92        -   LC-CDR3 having the amino acid sequence of SEQ ID NO:95.

8. The antigen-binding molecule according to any one of paras 1 to 6,wherein the antigen-binding molecule comprises:

-   -   (i) a heavy chain variable (VH) region incorporating the        following CDRs:        -   HC-CDR1 having the amino acid sequence of SEQ ID NO:41        -   HC-CDR2 having the amino acid sequence of SEQ ID NO:44        -   HC-CDR3 having the amino acid sequence of SEQ ID NO:47; and    -   (ii) a light chain variable (VL) region incorporating the        following CDRs:        -   LC-CDR1 having the amino acid sequence of SEQ ID NO:89        -   LC-CDR2 having the amino acid sequence of SEQ ID NO:92        -   LC-CDR3 having the amino acid sequence of SEQ ID NO:95.

9. The antigen-binding molecule according to any one of paras 1 to 6,wherein the antigen-binding molecule comprises:

-   -   (i) a heavy chain variable (VH) region incorporating the        following CDRs:        -   HC-CDR1 having the amino acid sequence of SEQ ID NO:41        -   HC-CDR2 having the amino acid sequence of SEQ ID NO:44        -   HC-CDR3 having the amino acid sequence of SEQ ID NO:47; and    -   (ii) a light chain variable (VL) region incorporating the        following CDRs:        -   LC-CDR1 having the amino acid sequence of SEQ ID NO:90        -   LC-CDR2 having the amino acid sequence of SEQ ID NO:92        -   LC-CDR3 having the amino acid sequence of SEQ ID NO:96.

10. The antigen-binding molecule according to any one of paras 1 to 6,wherein the antigen-binding molecule comprises;

-   -   (i) a heavy chain variable (VH) region incorporating the        following CDRs:        -   HC-CDR1 having the amino acid sequence of SEQ ID NO:41        -   HC-CDR2 having the amino acid sequence of SEQ ID NO:44        -   HC-CDR3 having the amino acid sequence of SEQ ID NO:47; and    -   (ii) a light chain variable (VL) region incorporating the        following CDRs:        -   LC-CDR1 having the amino acid sequence of SEQ ID NO:88        -   LC-CDR2 having the amino acid sequence of SEQ ID NO:92        -   LC-CDR3 having the amino acid sequence of SEQ ID NO:98.

11. The antigen-binding molecule according to any one of paras 1 to 6,wherein the antigen-binding molecule comprises:

-   -   (i) a heavy chain variable (VH) region incorporating the        following CDRs:        -   HC-CDR1 having the amino acid sequence of SEQ ID NO:41        -   HC-CDR2 having the amino acid sequence of SEQ ID NO:45        -   HC-CDR3 having the amino acid sequence of SEQ ID NO:47; and    -   (ii) a light chain variable (VL) region incorporating the        following CDRs:        -   LC-CDR1 having the amino acid sequence of SEQ ID NO:88        -   LC-CDR2 having the amino acid sequence of SEQ ID NO:93        -   LC-CDR3 having the amino acid sequence of SEQ ID NO:95.

12. The antigen-binding molecule according to any one of paras 1 to 6,wherein the antigen-binding molecule comprises:

-   -   (i) a heavy chain variable (VH) region incorporating the        following CDRs:        -   HC-CDR1 having the amino acid sequence of SEQ ID NO:41        -   HC-CDR2 having the amino acid sequence of SEQ ID NO:45        -   HC-CDR3 having the amino acid sequence of SEQ ID NO:49; and    -   (ii) a light chain variable (VL) region incorporating the        following CDRs:        -   LC-CDR1 having the amino acid sequence of SEQ ID NO:88        -   LC-CDR2 having the amino acid sequence of SEQ ID NO:93        -   LC-CDR3 having the amino acid sequence of SEQ ID NO:95.

13. The antigen-binding molecule according to any one of paras 1 to 6,wherein the antigen-binding molecule comprises:

-   -   (i) a heavy chain variable (VH) region incorporating the        following CDRs:        -   HC-CDR1 having the amino acid sequence of SEQ ID NO:41        -   HC-CDR2 having the amino acid sequence of SEQ ID NO:45        -   HC-CDR3 having the amino acid sequence of SEQ ID NO:50; and    -   (ii) a light chain variable (VL) region incorporating the        following CDRs:        -   LC-CDR1 having the amino acid sequence of SEQ ID NO:88        -   LC-CDR2 having the amino acid sequence of SEQ ID NO:93        -   LC-CDR3 having the amino acid sequence of SEQ ID NO:95.

14. The antigen-binding molecule according to any one of paras 1 to 6,wherein the antigen-binding molecule comprises:

-   -   (i) a heavy chain variable (VH) region incorporating the        following CDRs:        -   HC-CDR1 having the amino acid sequence of SEQ ID NO:41        -   HC-CDR2 having the amino acid sequence of SEQ ID NO:45        -   HC-CDR3 having the amino acid sequence of SEQ ID NO:48; and    -   (ii) a light chain variable (VL) region incorporating the        following CDRs:        -   LC-CDR1 having the amino acid sequence of SEQ ID NO:88        -   LC-CDR2 having the amino acid sequence of SEQ ID NO:92        -   LC-CDR3 having the amino acid sequence of SEQ ID NO:95.

15. The antigen-binding molecule according to any one of paras 1 to 6,wherein the antigen-binding molecule comprises:

-   -   (i) a heavy chain variable (VH) region incorporating the        following CDRs:        -   HC-CDR1 having the amino acid sequence of SEQ ID NO:41        -   HC-CDR2 having the amino acid sequence of SEQ ID NO:45        -   HC-CDR3 having the amino acid sequence of SEQ ID NO:48; and    -   (ii) a light chain variable (VL) region incorporating the        following CDRs:        -   LC-CDR1 having the amino acid sequence of SEQ ID NO:88        -   LC-CDR2 having the amino acid sequence of SEQ ID NO:92        -   LC-CDR3 having the amino acid sequence of SEQ ID NO:97.

16. The antigen-binding molecule according to any one of paras 1 to 6,wherein the antigen-binding molecule comprises:

-   -   (i) a heavy chain variable (VH) region incorporating the        following CDRs:        -   HC-CDR1 having the amino acid sequence of SEQ ID NO:42        -   HC-CDR2 having the amino acid sequence of SEQ ID NO:45        -   HC-CDR3 having the amino acid sequence of SEQ ID NO:48; and    -   (ii) a light chain variable (VL) region incorporating the        following CDRs:        -   LC-CDR1 having the amino acid sequence of SEQ ID NO:88        -   LC-CDR2 having the amino acid sequence of SEQ ID NO:92        -   LC-CDR3 having the amino acid sequence of SEQ ID NO:95.

17. The antigen-binding molecule according to any one of paras 1 to 5,wherein the antigen-binding molecule comprises:

-   -   (i) a heavy chain variable (VH) region incorporating the        following CDRs:        -   HC-CDR1 having the amino acid sequence of SEQ ID NO:158        -   HC-CDR2 having the amino acid sequence of SEQ ID NO:159        -   HC-CDR3 having the amino acid sequence of SEQ ID NO:160; and    -   (ii) a light chain variable (VL) region incorporating the        following CDRs:        -   LC-CDR1 having the amino acid sequence of SEQ ID NO:165        -   LC-CDR2 having the amino acid sequence of SEQ ID NO:166        -   LC-CDR3 having the amino acid sequence of SEQ ID NO:167.

18. The antigen-binding molecule according to any one of paras 1 to 4,wherein the antigen-binding molecule is capable of binding to apolypeptide comprising the amino acid sequence of SEQ ID NO:22.

19. The antigen-binding molecule according to any one of paras 1 to 4 orpara 18 wherein the antigen-binding molecule comprises:

-   -   (i) a heavy chain variable (VH) region incorporating the        following CDRs:        -   HC-CDR1 having the amino acid sequence of SEQ ID NO:128        -   HC-CDR2 having the amino acid sequence of SEQ ID NO:129        -   HC-CDR3 having the amino acid sequence of SEQ ID NO:130; and    -   (ii) a light chain variable (VL) region incorporating the        following CDRs:        -   LC-CDR1 having the amino acid sequence of SEQ ID NO:136        -   LC-CDR2 having the amino acid sequence of SEQ ID NO:137        -   LC-CDR3 having the amino acid sequence of SEQ ID NO:138.

20. The antigen-binding molecule according to any one of paras 1 to 4 orpara 18 wherein the antigen-binding molecule comprises:

-   -   (i) a heavy chain variable (VH) region incorporating the        following CDRs:        -   HC-CDR1 having the amino acid sequence of SEQ ID NO:144        -   HC-CDR2 having the amino acid sequence of SEQ ID NO:145        -   HC-CDR3 having the amino acid sequence of SEQ ID NO:146; and    -   (ii) a light chain variable (VL) region incorporating the        following CDRs:        -   LC-CDR1 having the amino acid sequence of SEQ ID NO:151        -   LC-CDR2 having the amino acid sequence of SEQ ID NO:152        -   LC-CDR3 having the amino acid sequence of SEQ ID NO:153.

21. The antigen-binding molecule according to any one of paras 1 to 4,wherein the antigen-binding molecule comprises:

-   -   (i) a VH region comprising an amino acid sequence having at        least 70% sequence identity to the amino acid sequence of SEQ ID        NO:24; and        -   a VL region comprising an amino acid sequence having at            least 70% sequence identity to the amino acid sequence of            SEQ ID NO:74;            or    -   (ii) a VH region comprising an amino acid sequence having at        least 70% sequence identity to the amino acid sequence of SEQ ID        NO:25; and        -   a VL region comprising an amino acid sequence having at            least 70% sequence identity to the amino acid sequence of            SEQ ID NO:75;            or    -   (iii) a VH region comprising an amino acid sequence having at        least 70% sequence identity to the amino acid sequence of SEQ ID        NO:26; and        -   a VL region comprising an amino acid sequence having at            least 70% sequence identity to the amino acid sequence of            SEQ ID NO:76;            or    -   (iv) a VH region comprising an amino acid sequence having at        least 70% sequence identity to the amino acid sequence of SEQ ID        NO:27; and        -   a VL region comprising an amino acid sequence having at            least 70% sequence identity to the amino acid sequence of            SEQ ID NO:77;            or    -   (v) a VH region comprising an amino acid sequence having at        least 70% sequence identity to the amino acid sequence of SEQ ID        NO:28; and        -   a VL region comprising an amino acid sequence having at            least 70% sequence identity to the amino acid sequence of            SEQ ID NO:78;            or    -   (vi) a VH region comprising an amino acid sequence having at        least 70% sequence identity to the amino acid sequence of SEQ ID        NO:29; and        -   a VL region comprising an amino acid sequence having at            least 70% sequence identity to the amino acid sequence of            SEQ ID NO:78;            or    -   (vii) a VH region comprising an amino acid sequence having at        least 70% sequence identity to the amino acid sequence of SEQ ID        NO:30; and        -   a VL region comprising an amino acid sequence having at            least 70% sequence identity to the amino acid sequence of            SEQ ID NO:78;            or    -   (viii) a VH region comprising an amino acid sequence having at        least 70% sequence identity to the amino acid sequence of SEQ ID        NO:31; and        -   a VL region comprising an amino acid sequence having at            least 70% sequence identity to the amino acid sequence of            SEQ ID NO:79;            or    -   (ix) a VH region comprising an amino acid sequence having at        least 70% sequence identity to the amino acid sequence of SEQ ID        NO:32; and        -   a VL region comprising an amino acid sequence having at            least 70% sequence identity to the amino acid sequence of            SEQ ID NO:79;            or    -   (x) a VH region comprising an amino acid sequence having at        least 70% sequence identity to the amino acid sequence of SEQ ID        NO:33; and        -   a VL region comprising an amino acid sequence having at            least 70% sequence identity to the amino acid sequence of            SEQ ID NO:80;            or    -   (xi) a VH region comprising an amino acid sequence having at        least 70% sequence identity to the amino acid sequence of SEQ ID        NO:34; and        -   a VL region comprising an amino acid sequence having at            least 70% sequence identity to the amino acid sequence of            SEQ ID NO:81;            or    -   (xii) a VH region comprising an amino acid sequence having at        least 70% sequence identity to the amino acid sequence of SEQ ID        NO:35; and        -   a VL region comprising an amino acid sequence having at            least 70% sequence identity to the amino acid sequence of            SEQ ID NO:82;            or    -   (xiii) a VH region comprising an amino acid sequence having at        least 70% sequence identity to the amino acid sequence of SEQ ID        NO:36; and        -   a VL region comprising an amino acid sequence having at            least 70% sequence identity to the amino acid sequence of            SEQ ID NO:83;            or    -   (xiv) a VH region comprising an amino acid sequence having at        least 70% sequence identity to the amino acid sequence of SEQ ID        NO:37; and        -   a VL region comprising an amino acid sequence having at            least 70% sequence identity to the amino acid sequence of            SEQ ID NO:84;            or    -   (xv) a VH region comprising an amino acid sequence having at        least 70% sequence identity to the amino acid sequence of SEQ ID        NO:38; and        -   a VL region comprising an amino acid sequence having at            least 70% sequence identity to the amino acid sequence of            SEQ ID NO:85;            or    -   (xvi) a VH region comprising an amino acid sequence having at        least 70% sequence identity to the amino acid sequence of SEQ ID        NO:39; and        -   a VL region comprising an amino acid sequence having at            least 70% sequence identity to the amino acid sequence of            SEQ ID NO:86;            or    -   (xvii) a VH region comprising an amino acid sequence having at        least 70% sequence identity to the amino acid sequence of SEQ ID        NO:40; and        -   a VL region comprising an amino acid sequence having at            least 70% sequence identity to the amino acid sequence of            SEQ ID NO:87;            or    -   (xviii) a VH region comprising an amino acid sequence having at        least 70% sequence identity to the amino acid sequence of SEQ ID        NO:127; and        -   a VL region comprising an amino acid sequence having at            least 70% sequence identity to the amino acid sequence of            SEQ ID NO:135;            or    -   (xix) a VH region comprising an amino acid sequence having at        least 70% sequence identity to the amino acid sequence of SEQ ID        NO:143; and        -   a VL region comprising an amino acid sequence having at            least 70% sequence identity to the amino acid sequence of            SEQ ID NO:150;            or    -   (xx) a VH region comprising an amino acid sequence having at        least 70% sequence identity to the amino acid sequence of SEQ ID        NO:157; and        -   a VL region comprising an amino acid sequence having at            least 70% sequence identity to the amino acid sequence of            SEQ ID NO:164.

22. The antigen-binding molecule according to any one of paras 1 to 21,wherein the antigen-binding molecule is capable of binding to human HER3and one or more of mouse HER3, rat HER3 and cynomolgous macaque HER3.

23. An antigen-binding molecule, optionally isolated, comprising (i) anantigen-binding molecule according to any one of paras 1 to 22, and (ii)an antigen-binding molecule capable of binding to an antigen other thanHER3.

24. The antigen-binding molecule according to any one of paras 1 to 23,wherein the antigen-binding molecule is capable of binding to cellsexpressing HER3 at the cell surface.

25. The antigen-binding molecule according to any one of paras 1 to 24,wherein the antigen-binding molecule is capable of inhibitingHER3-mediated signalling.

26. The antigen-binding molecule according to any one of paras 1 to 25,wherein the antigen-binding molecule comprises an Fc region, the Fcregion comprising a polypeptide having: (i) C at the positioncorresponding to position 242, and C at the position corresponding toposition 334, and (ii) one or more of: A at the position correspondingto position 236, at the position corresponding to position 239, E at theposition corresponding to position 332, L at the position correspondingto position 330, K at the position corresponding to position 345, and Gat the position corresponding to position 430.

27. The antigen-binding molecule according to para 26 wherein the Fcregion comprises a polypeptide having C at the position corresponding toposition 242, C at the position corresponding to position 334, A at theposition corresponding to position 236, D at the position correspondingto position 239, E at the position corresponding to position 332, and Lat the position corresponding to position 330.

28. A chimeric antigen receptor (CAR) comprising an antigen-bindingmolecule according to any one of paras 1 to 27.

29. A nucleic add, or a plurality of nucleic adds, optionally isolated,encoding an antigen-binding molecule according to any one of paras 1 to27 or a CAR according to para 28.

30. An expression vector, or a plurality of expression vectors,comprising a nucleic add or a plurality of nucleic adds according topara 29.

31. A cell comprising an antigen-binding molecule according to any oneof paras 1 to 27, a CAR according to para 28, a nucleic add or aplurality of nucleic adds according to para 29, or an expression vectoror a plurality of expression vectors according to para 30.

32. A method comprising culturing a cell comprising a nucleic add or aplurality of nucleic adds according to para 29, or an expression vectoror a plurality of expression vectors according to para 30, underconditions suitable for expression of the antigen-binding molecule orCAR from the nucleic acid(s) or expression vector(s).

33. A composition comprising an antigen-binding molecule according toany one of paras 1 to 27, a CAR according to para 28, a nucleic acid ora plurality of nucleic acids according to para 29, an expression vectoror a plurality of expression vectors according to para 30, or a cellaccording to para 31.

34. An antigen-binding molecule according to any one of paras 1 to 27, aCAR according to para 28, a nucleic acid or a plurality of nucleic acidsaccording to para 29, an expression vector or a plurality of expressionvectors according to para 30, a cell according to para 31, or acomposition according to para 33 for use in a method of medicaltreatment or prophylaxis.

35. An antigen-binding molecule according to any one of paras 1 to 27, aCAR according to para 28, a nucleic acid or a plurality of nucleic acidsaccording to para 29, an expression vector or a plurality of expressionvectors according to para 30, a cell according to para 31, or acomposition according to para 33, for use in a method of treatment orprevention of a cancer.

36. Use of an antigen-binding molecule according to any one of paras 1to 27, a CAR according to para 28, a nucleic acid or a plurality ofnucleic acids according to para 29, an expression vector or a pluralityof expression vectors according to para 30, a cell according to para 31,or a composition according to para 33, in the manufacture of amedicament for use in a method of treatment or prevention of a cancer.

37. A method of treating or preventing a cancer, comprisingadministering to a subject a therapeutically or prophylacticallyeffective amount of an antigen-binding molecule according to any one ofparas 1 to 27, a CAR according to para 28, a nucleic acid or a pluralityof nucleic acids according to para 29, an expression vector or aplurality of expression vectors according to para 30, a cell accordingto para 31, or a composition according to para 33.

38. The antigen-binding molecule, CAR, nucleic acid or plurality ofnucleic acids, expression vector or plurality of expression vectors,cell or composition for use according to para 34 or para 35, the useaccording to para 36 or the method according to para 37, wherein themethod additionally comprises administration of an inhibitor ofsignalling mediated by an EGFR family member, optionally wherein theinhibitor of signalling mediated by an EGFR family member is aninhibitor of signalling mediated by HER2 and/or EGFR.

39. The antigen-binding molecule, CAR, nucleic: acid or plurality ofnucleic acids, expression vector or plurality of expression vectors,cell or composition for use, the use or the method according to any oneof paras 34 to para 38, wherein the cancer is selected from: a cancercomprising cells expressing an EGFR family member, a cancer comprisingcells expressing HER3, a solid tumor, breast cancer, breast carcinoma,ductal carcinoma, gastric cancer, gastric carcinoma, gastricadenocarcinoma, colorectal cancer, colorectal carcinoma, colorectaladenocarcinoma, head and neck cancer, squamous carcinoma of the head andneck (SCCHN), lung cancer, lung adenocarcinoma, squamous cell lungcarcinoma, ovarian cancer, ovarian carcinoma, ovarian serousadenocarcinoma, kidney cancer, renal cell carcinoma, renal clear cellcarcinoma, renal cell adenocarcinoma, renal papillary cell carcinoma,pancreatic cancer, pancreatic adenocarcinoma, pancreatic ductaladenocarcinoma, cervical cancer, cervical squamous cell carcinoma, skincancer, melanoma, esophageal cancer, esophageal adenocarcinoma, livercancer, hepatocellular carcinoma, cholangiocarcinoma, uterine cancer,uterine corpus endometrial carcinoma, thyroid cancer, thyroid carcinoma,pheochromocytoma, paraganglioma, bladder cancer, bladder urothelialcarcinoma, prostate cancer, prostate adenocarcinoma, sarcoma andthymoma.

40. A method of inhibiting HER3-mediated signalling, comprisingcontacting HER3-expressing cells with an antigen-binding moleculeaccording to any one of paras 1 to 27.

41. A method of reducing the number or activity of HER3-expressingcells, the method comprising contacting HER3-expressing cells with anantigen-binding molecule according to any one of paras 1 to 27.

42. An in vitro complex, optionally isolated, comprising anantigen-binding molecule according to any one of paras 1 to 27 bound toHER3.

43. A method comprising contacting a sample containing, or suspected tocontain, HER3 with an antigen-binding molecule according to any one ofparas 1 to 27, and detecting the formation of a complex of theantigen-binding molecule with HER3.

44. A method of selecting or stratifying a subject for treatment with aHER3-targeted agent, the method comprising contacting, in vitro, asample from the subject with an antigen-binding molecule according toany one of paras 1 to 27 and detecting the formation of a complex of theantigen-binding molecule with HER3.

45. Use of an antigen-binding molecule according to any one of paras 1to 27 as an in vitro or in vivo diagnostic or prognostic agent.

46. Use of an antigen-binding molecule according to any one of paras 1to 27 in a method for detecting, localizing or imaging a cancer,optionally wherein the cancer is selected from: a cancer comprisingcells expressing an EGFR family member, a cancer comprising cellsexpressing HER3, a solid tumor, breast cancer, breast carcinoma, ductalcarcinoma, gastric cancer, gastric carcinoma, gastric adenocarcinoma,colorectal cancer, colorectal carcinoma, colorectal adenocarcinoma, headand neck cancer, squamous cell carcinoma of the head and neck (SCCHN),lung cancer, lung adenocarcinoma, squamous cell lung carcinoma, ovariancancer, ovarian carcinoma, ovarian serous adenocarcinoma, kidney cancer,renal cell carcinoma, renal dear cell carcinoma, renal celladenocarcinoma, renal papillary cell carcinoma, pancreatic cancer,pancreatic adenocarcinoma, pancreatic ductal adenocarcinoma, cervicalcancer, cervical squamous cell carcinoma, skin cancer, melanoma,esophageal cancer, esophageal adenocarcinoma, liver cancer,hepatocellular carcinoma, cholangiocarcinoma, uterine cancer, uterinecorpus endometrial carcinoma, thyroid cancer, thyroid carcinoma,pheochromocytoma, paraganglioma, bladder cancer, bladder urothelialcarcinoma, prostate cancer, prostate adenocarcinoma, sarcoma andthymoma.

The invention includes the combination of the aspects and preferredfeatures described except where such a combination is clearlyimpermissible or expressly avoided.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.

Aspects and embodiments of the present invention will now beillustrated, by way of example, with reference to the accompanyingfigures. Further aspects and embodiments will be apparent to thoseskilled in the art. All documents mentioned in this text areincorporated herein by reference.

Throughout this specification, including the claims which follow, unlessthe context requires otherwise, the word “comprise,” and variations suchas “comprises” and “comprising,” will be understood to imply theinclusion of a stated integer or step or group of integers or steps butnot the exclusion of any other integer or step or group of integers orsteps.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a” “an,” and “the” include plural referentsunless the context clearly dictates otherwise. Ranges may be expressedherein as from “about” one particular value, and/or to “about” anotherparticular value. When such a range is expressed, another embodimentincludes from the one particular value and/or to the other particularvalue. Similarly, when values are expressed as approximations, by theuse of the antecedent “about,” it will be understood that the particularvalue forms another embodiment.

Where a nucleic acid sequence is disclosed herein, the reversecomplement thereof is also expressly contemplated.

Methods described herein may preferably performed in vitro. The term “invitro” is intended to encompass procedures performed with cells inculture whereas the term “in vivo” is intended to encompass procedureswith/on intact multi-cellular organisms.

BRIEF DESCRIPTION OF THE FIGURES

Embodiments and experiments illustrating the principles of the inventionwill now be discussed with reference to the accompanying figures.

FIGS. 1A and 1B. Histograms showing staining of cells by anti-HER3antibodies as determined by flow cytometry. Histograms show staining ofHEK293 cells (which do not express HER3), or HEK293 HER3 overexpressingcells (HEK293 HER3 O/E) by (1A, 1B) anti-HER3 antibody clone 10D1 and(1B) anti-HER3 antibody clone LJM716.

FIGS. 2A and 2B. Histograms showing staining of cells by anti-HER3antibodies as determined by flow cytometry. Histograms show staining ofHEK293 cells (which do not express HER3), or HEK293 HER3 overexpressingcells (HEK293 HER3 O/E) by (2A, 2B) anti-HER3 antibody clone 4-35-B2 and(2B) anti-HER3 antibody clone LJM716.

FIGS. 3A and 3B. Histograms showing staining of cells by anti-HER3antibodies as determined by flow cytometry. Histograms show staining ofHEK293 cells (which do not express HER3), or HEK293 HER3 overexpressingcells (HEK293 HER3 O/E) by (3A, 3B) anti-HER3 antibody clone 4-35-B4 and(3B) anti-HER3 antibody clone LJM716.

FIG. 4 . Histograms showing staining of cells by anti-HER3 antibodies asdetermined by flow cytometry. Histograms show staining of HEK293 cells(which do not express HER3), or HEK293 HER3 overexpressing cells (HEK293HER3 O/E) by anti-HER3 antibody clone 10A6.

FIGS. 5A and 5B. Graphs showing the results of ELISA analysis of bindingof anti-HER3 antibody clone 10D1 to (5A) human, mouse, rat andcynomolgus macaque HER3, and (5B) human EGFR and human HER2. EC₅₀ valuesare shown.

FIGS. 6A and 6B. Graphs showing the results of ELISA analysis of bindingof anti-HER3 antibody clone 4-35-B2 to (6A) human, mouse, rat andcynomolgus macaque HER3, and (6B) human EGFR and human HER2.

FIGS. 7A and 7B. Graphs showing the results of ELISA analysis of bindingof anti-HER3 antibody cone 4-35-B4 to (7A) human HER3, human EGFR andhuman HER2, and (7B) human, mouse, rat and cynomolgus macaque HER3.

FIG. 8 . Representative sensorgram showing the results of analysis ofaffinity of binding of anti-HER3 antibody clone 10D1 to human HER3. Kon,Koff and K_(D) are shown.

FIG. 9 . Representative sensorgram showing the results of analysis ofaffinity of binding of anti-HER3 antibody clone 4-35-B2 to human HER3.

FIG. 10 . Representative sensorgram showing the results of analysis ofaffinity of binding of anti-HER3 antibody clone 4-35-B4 to human HER3.

FIG. 11 . Graph showing the results of analysis of stability ofanti-HER3 antibody clone 10D1 by Differential Scanning Fluorimetryanalysis.

FIG. 12 . Graph showing the results of the analysis ofrecombinantly-expressed anti-HER3 antibody clone 10D1 by size exclusionchromatography.

FIG. 13 . Images showing the results of the analysis of anti-HER3antibody clone 10D1 expression by SDS-PAGE and western blot. Lanes:M1=TaKaRa protein marker Cat. No. 3452; M2=GenScript protein marker Cat.No. M00521; 1=reducing conditions; 2=non-reducing conditions; P=positivecontrol: human IgG1, Kappa (Sigma Cat. No. I5154). For western blot, theprimary antibodies used were goat anti-human IgG-HRP (GenScript Cat No.A00166) and goat anti-human kappa-HRP (SouterhnBiotech Cat No. 2060-05).

FIGS. 14A and 14B. Representative sensorgram and table showing theresults of analysis of competition between different anti-HER3 antibodyclones for binding to HER3.

FIG. 15 . Graph showing the results of analysis of the inhibition ofinteraction between HER3 and HER2 by anti-HER3 antibody clone 10D1 asdetermined by ELISA.

FIGS. 16A and 16B. Table and histograms showing gene and proteinexpression of EGFR protein family members and their ligands by differentcancer cell lines.

FIG. 17 . Images showing the results of analysis of the effect ofanti-HER3 antibody clone 10D1 treatment on the HER3-mediated signallingin N87 and FaDu cells by phospho-western blot. UN=untreated; T=treatedwith anti-HER3 antibody clone 10D1.

FIG. 18 . Images and graph showing the results of analysis of the effectof anti-HER3 antibody clone 10D1 treatment on the HER3-mediatedsignalling in FaDu cells using the Phosphoprotein Antibody Array assaykit. Untreated=untreated FaDu cells; Treated=FaDu cells treated withanti-HER3 antibody clone 10D1.

FIGS. 19A and 19B. Graphs showing the percent confluence of cellsrelative to an untreated control condition (100%), for the indicatedcells lines as determined by CCK8 assay, following incubation in thepresence of anti-HER3 antibody clone 10D1. (19A) Shows the resultsobtained for N87 cells, and (19B) shows the results obtained for FaDucells.

FIG. 20 . Graph showing the results of analysis of tumour volume overtime in a N87 cell-line derived mouse gastric carcinoma model. Anti-HER3antibody clone 10D1 was administered IP, biweekly at 500 μg per dose fora total of 10 doses. A control treatment group received an equal volumeof PBS (vehicle).

FIG. 21 . Graph showing the results of analysis of tumour volume overtime in a N87 cell-line derived mouse gastric carcinoma model. Anti-HER3antibody clone 4-35-B2 was administered IP, weekly at 11 mg/kg per dosefor a total of 4 doses. A control treatment group received an equalamount of isotype control antibody (isotype).

FIG. 22 . Graph showing the results of analysis of tumour volume overtime in a SNU16 cell-line derived mouse gastric carcinoma model.Anti-HER3 antibody clone 10D1 was administered IP, biweekly at 500 μgper dose for a total of 9 doses. A control treatment group received anequal volume of PBS (vehicle).

FIG. 23 , Graph showing the results of analysis of tumour volume overtime in a FaDu cell-line derived mouse model of head and neck squamouscell carcinoma. Anti-HER3 antibody clone 10D1 was administered IP,weekly at 500 μg per dose for a total of 4 doses. Control treatmentgroups received an equal volume of PBS (vehicle), or the same dose of anisotype control antibody (isotype).

FIG. 24 . Graph showing the results of analysis of tumour volume overtime in a FaDu cell-line derived mouse model of head and neck squamouscell carcinoma. Anti-HER3 antibody clone 10D1 was administered IP,biweekly at 500 μg per dose for a total of 8 doses. A control treatmentgroup received an equal volume of PBS (vehicle).

FIG. 25 . Graph showing the results of analysis of tumour volume overtime in an OvCAR8 cell-line derived mouse model of ovarian carcinoma.Anti-HER3 antibody clone 10D1 was administered IP, biweekly at 500 μgper dose for a total of 9 doses. A control treatment group received anequal volume of PBS (vehicle).

FIG. 26 . Graph showing the results of analysis of tumour volume overtime in a HCC-95 cell-line derived mouse model of squamous king cellcarcinoma. Anti-HER3 antibody clone 10D1 was administered IP, biweeklyat 500 μg per dose for a total of 4 doses. A control treatment groupreceived an equal volume of PBS (vehicle).

FIG. 27 . Graph showing the results of analysis of tumour volume overtime in an A549 cell-line derived mouse model of lung adenocarcinoma.Anti-HER3 antibody clone 10D1 was administered IP, biweekly at 500 μgper dose for a total of 10 doses. A control treatment group received anequal volume of PBS (vehicle).

FIG. 28 . Graph showing the results of analysis of tumour volume overtime in an A549 cell-line derived mouse model of lung adenocarcinoma.Anti-HER3 antibody clone 4-35-B2 was administered IP, biweekly at 500 μgper dose for a total of 4 doses. A control treatment group received anequal volume of PBS (vehicle).

FIG. 29 . Graph showing the results of analysis of tumour volume overtime in an ACHN cell-line derived mouse model of renal cell carcinoma.Anti-HER3 antibody clone 10D1 was administered IP, biweekly at 500 μgper dose for a total of 7 doses. A control treatment group received anequal volume of PBS (vehicle).

FIG. 30 . Histogram showing staining of cells by anti-HER3 antibodyclone 10D1_c89 as determined by flow cytometry. Histograms show stainingof HEK293 cells (which do not express HER3), or HEK293 HER3overexpressing cells (HEK293 HER3 O/E).

FIG. 31 . Histogram showing staining of cells by anti-HER3 antibodyclone 10D1_c90 as determined by flow cytometry. Histograms show stainingof HEK293 cells (which do not express HER3), or HEK293 HER3overexpressing cells (HEK293 HER3 O/E).

FIG. 32 . Histogram showing staining of cells by anti-HER3 antibodyclone 10D1_c91 as determined by flow cytometry. Histograms show stainingof HEK293 cells (which do not express HER3), or HEK293 HER3overexpressing cells (HEK293 HER3 O/E).

FIGS. 33A and 33B. Graphs showing the results of ELISA analysis ofbinding of anti-HER3 antibody 10D1 variant clones to human HER3. (33A)shows binding of anti-HER3 antibody clones 10D1 (referred to as 10D1P),10D1_c75, 10D1_c76, 10D1_c77, 10D1_c78, 10D1_11B (referred to asv11b78L), 10D1_c85, 10D1_c85o1, 10D1_c85o2, 10D1_c87, 10D1_c89,10D1_c90, 10D1_c91, 10D1_c93, LJM716 and hIgG (negative control). (33B)shows the same data as 33A, but for clones 10D1_c89, 10D1_c90, 10D1_c91and LJM716 only.

FIGS. 34A and 34B. Graphs showing the results of the analysis ofrecombinantly-expressed anti-HER3 antibody 10D1 variant clones by sizeexclusion chromatography. (34A) shows results for anti-HER3 antibodyclones 10D1_c93, 10D1_c75, 10D1_c76, 10D1_c77, 10D1_c78, 10D1_11B(referred to as C78 v11b), 10D1_c85, 10D1_c85o1, 10D1_c85o2, 10D1_c89,10D1_c90, 10D1_c91 and 10D1_c93. (34B) shows the same data as 33A, butfor clones 10D1_c89, 10D1_c90, 10D1_c91 and only.

FIGS. 35A to 35C. Graphs showing the results analysis of stability ofanti-HER3 antibody 10D1 variant clones by Differential ScanningFluorimetry analysis. (35A) shows results for anti-HER3 antibody clonesLJM716 (also referred to as Elgemtumab), 10D1 (referred to as 10D1(parental)), 10D1_c75, 10D1_c76, 10D1_c77 and 10D1_c75. (35B) showsresults for 10D1_c85o2, 10D1_c87, 10D1_c89, 10D1_11B (referred to asc78_V11B), 10D1_c85 and 10D1_c85o1. (35C) shows results for 10D1_c90,10D1_c91 and 10D1_c93.

FIGS. 36A to 36M. Representative sensorgrams showing the results ofanalysis of affinity of anti-HER3 antibody 10D1 variant clones to humanHER3. Kon, Koff and K_(D) are shown. (36A) shows results for clone10D1_c89, (36B) shows results for clone 10D1_c90, (36C) shows resultsfor clone 10D1_c91, (36D) shows results for clone 10D1_c11B, (36E) showsresults for clone 10D1_c85o2, (36F) shows results for clone 10D1_c87,(36G) shows results for clone 10D1_c93, (36H) shows results for clone10D1_c76, (36I) shows results for clone 10D1_c77, (36J) shows resultsfor clone 10D1_c78, (36K) shows results for clone 10D1_c75, (36L) showsresults for clone 10D1_c55, and (36M) shows results for clone10D1_c85o1.

FIG. 37 . Table summarising properties of anti-HER3 antibody 10D1variant clones relevant to safety and developability.

FIGS. 38A and 38B. Bio-Layer Interferometry (38A) and thermostability(38B) analysis of Fc-modified anti-HER3 antibody clone 10D1 comprisingGASDALIE and LCKC substitutions in CH2 region. (38A) BLI shows arepresentative sensorgram showing the results of analysis of affinity ofbinding to FcγRIIIa by Fc-modified anti-HER3 antibody clone 10D1. Kon,Koff and K_(D) are shown.

FIGS. 39A and 39B. Representative sensorgrams showing results ofanalysis of affinity of binding to FcγRIIIa by (39A) non-Fc-modifiedanti-HER3 antibody clone 10D1 and (39B) Fc-modified anti-HER3 antibodyclone 10D1 comprising GASD substitutions in CH2 region. Kon, Koff andK_(D) are shown.

FIG. 40 . Graph showing the results of analysis of stability ofanti-HER3 antibody alone 10D1 GASD variant by Differential ScanningFluorimetry analysis.

FIGS. 41A and 41B. Tables showing the binding affinity for mouse andhuman Fc receptors of anti-HER3 antibody clones 10D1F.FcA and 10D1F.FcB(GASDALIE-LCKC variant) compared to silent variant N297Q, isoformvariants, and commercially available antibodies. ND=K_(D) Not Determineddue to low binding affinity.

FIGS. 42A and 42B. Histograms showing staining of cells by anti-HER3antibodies as determined by flow cytometry. Histograms show staining ofHEK293 cells (which do not express HER3), or HEK293 HER3 overexpressingcells (HEK293 HER3 O/E) by (42A) anti-HER3 antibody clone 10D1F.FcA and(42B) anti-HER3 antibody clones 10D1 and LJM-716.

FIG. 43 . Graph showing the results of ELISA analysis of binding ofanti-HER3 antibody clone 10D1F.FcA to human EGFR (HER1) and human HER2.EC₅₀ values are shown.

FIG. 44 . Histogram showing staining of cells by anti-HER3 and anti-HER4antibodies as determined by flow cytometry. Histogram shows staining ofHEK293 HER4 overexpressing cells by anti-HER3 antibody clone 10D1F.FcA,anti-HER3 antibodies LJM-716 and MM-121, and commercial anti-HER4antibody.

FIG. 45 . Graph showing the results of ELISA analysis of binding ofanti-HER3 antibody clone 10D1F.FcA to human, mouse, rat and cynomolgusmacaque HER3. EC₅₀ values are shown.

FIGS. 46A and 46B. Representative sensorgrams showing the results ofanalysis of affinity of binding of anti-HER3 antibody clones (46A)10D1F.FcA and (46B) 10D1F.FcB to human HER3. Kon, Koff and K_(D) areshown.

FIGS. 47A and 47B. Graph showing the results of analysis of stability ofanti-HER3 antibody clones (47A) 10D1F.FcA and (47B) 10D1F.FcB byDifferential Scanning Fluorimetry analysis.

FIGS. 48A and 48B. Graph showing the results of purity analysis ofanti-HER3 antibody clones (48A) 10D1F.FcA and (48B) 10D1F.FcB by sizeexclusion chromatography.

FIGS. 49A and 49B. Representative sensorgram and table showing theresults of analysis of competition for binding to HER3 between anti-HER3antibody clone 10D1F.FcA and anti-HER3 antibodies M-05-74 and M-08-11.

FIG. 50 . Graph and tables showing the results of pharmacokineticanalysis of anti-HER3 antibody clone 10D1 in mice.

FIGS. 51A to 51F. Graphs showing the effect of anti-HER3 antibody clone10D1 treatment on blood cell counts (51A), electrolyte indices (51B) andindices of hepatoxicity, nephrotoxicity and pancreatic toxicity(51C-51F) in mice. Left bars represent vehicle control, right barsrepresent 10D1 treatment. Dotted lines indicate the end points of theCharles River reference range. Indices of hepatoxicity, nephrotoxicityand pancreatic toxicity include alanine aminotransferase (ALT),aspartate transaminase (AST), blood urea nitrogen (BUN), creatinine(CREA), alkaline phosphatase (ALP), glucose (GLU), calcium (CAL), totalbilirubin (BIL), total protein (TPR) and albumin (ALB).

FIG. 52 . Graph and table showing the results of analysis of theinhibition of interaction between HER2 and HER3 by anti-HER3 antibodyclone 10D1F.FcA and antibodies MM-121, LJM-716 and Roche M05 asdetermined by ELISA.

FIG. 53 . Graph and table showing the results of analysis of theinhibition of interaction between EGFR and HER3 by anti-HER3 antibodyclone 10D1F.FcA and antibodies MM-121 and LJM716, as determined byELISA.

FIG. 54 . Graph and table showing the results of analysis of the abilityof anti-HER3 antibody clones 10D1F.FcA (10D1F.A), 10D1F.FcB (10D1F.B),10D1F-hIgG1 (N297Q) and anti-HER3 antibodies LJM-716 and Seribantumab(MM-121), to induce antibody-dependent cell-mediated cytotoxicity(ADCC). EC₅₀ values are shown.

FIGS. 55A to 55C. Images showing the results of analysis of the effectof anti-HER3 antibody treatment on HER3-mediated signalling in (55A)N87, (55B) FaDu and (55C) OvCar8 cells by phospho-western blot.

FIGS. 56A and 56B. Graph and tables showing the results ofpharmacokinetic analysis of anti-HER3 antibody clones (56A) 10D1F.FcAand (56B) 10D1F.FcB in mice. Parameters: maximum concentration(C_(max)), T_(max), AUC (0-336 hr), AUC (0-infinity), Half-life(t_(1/2)), Clearance (CL), Volume of distribution at steady state(V_(ss)).

FIGS. 57A to 57D. Graph and tables showing the results ofpharmacokinetic analysis of anti-HER3 antibody clones 10D1F.FcA and10D1F.FcB at (57A) 10 mg/kg, (57B) 25 mg/kg, (57C) 100 mg/kg and (57D)250 mg/kg in rats. Parameters: maximum concentration (C_(max)), T_(max),AUC (0-336 hr), AUC (0-infinity), Half-life (t_(1/2)), Clearance (CL),Volume of distribution at steady state (V_(ss)).

FIGS. 58A to 58F. Graphs showing the effect of treatment of anti-HER3antibody clone 10D1F.FcA or 10D1F.FcB at 200 ug (˜10 mg/kg), 500 ug (˜25mg/kg), 2 mg (˜100 mg/kg), or 5 mg (˜250 mg/kg) on (58A, 58B) red bloodcell indices, (58C) white blood cell indices, (58D) hepatotoxicity,(58E) kidney and pancreatic indices, and (58F) electrolyte indices.

FIGS. 59A to 59D. Graphs showing the effect of treatment anti-HER3antibody clone 10D1F.FcA on percentage tumour inhibition in in vitromouse cancer models using N87 cells (gastric cancer), HCC95 cells (lungcancer), FaDu cells (head and neck cancer), SNU-16 cells (gastriccancer), A549 cells (lung cancer), OvCar8 cells (ovarian cancer), ACHNcells (kidney cancer) and HT29 cells (colorectal cancer) in comparisonto (59A & 59B) anti-HER3 antibodies seribantumab and LJM-716 and (59C &59D) EGFR family therapies cetuximab, trastuzumab and pertuzumab.

FIG. 60 . Graph showing the results of analysis of tumour volume overtime in an A549 cell-line derived mouse model of lung adenocarcinomaafter biweekly treatment with the indicated concentrations of antibodiesfor six weeks (n=6, vehicle control n=8). Antibody administration isindicated by triangles along x-axis.

FIG. 61 . Graph showing the results of analysis of tumour volume overtime in a FaDu cell-line derived mouse model of head and neck squamouscell carcinoma after weekly treatment with the indicated concentrationsof antibodies for six weeks (n=6). Antibody administration is indicatedby triangles along x-axis.

FIG. 62 . Graph showing the results of analysis of tumour volume overtime in a OvCAR8 cell-line derived mouse model of ovarian carcinomaafter weekly treatment with the indicated concentrations of antibodiesfor six weeks (n=6). Antibody administration is indicated by trianglesalong x-axis.

FIGS. 63A to 63D. Box plots showing the results of analysis of pathwayactivation by gene set enrichment analysis, for cancer cell linestreated with 10D1F.FcA, LJM-716 or seribantumab in in vitrophosphorylation assays. 63A shows the results obtained from N87 cells,63B shows the results obtained from A549 cells, 63C shows the resultsobtained from OvCar8 cells and 63D shows the results obtained from FaDucells.

FIG. 64 . Images showing the results of analysis of the effect ofanti-HER3 antibody treatment on HER3-mediated signalling in A549 cellsby phospho-western blot, at the indicated time points.

FIG. 65 . Graph showing the results of analysis of inhibiting ofHER2:HER3 interaction by 10D1F.FcA or pertuzumab, as determined byPathHunter Pertuzumab Bioassay. IC50 (M) values are shown.

FIGS. 66A to 66C. Histograms showing the results of analysis ofexpression of EGFR, HER2 and HER3 by (66A) BCPAP (66B) BHT101 and (66C)SW1736 cells. 1=unstained cells, 2=isotype control, 3=cetuximab,4=trastuzumab, and 5=10D1F.FcA.

FIGS. 67A to 67C. Graphs showing the results of analysis of the abilityof different anti-ErbB antibodies to inhibit proliferation ofBRAF^(V600E) mutant thyroid cancer cell lines in vitro. 67A shows theresults obtained for BHT101 cells, 687 shows the results obtained forBCPAP cells, and 67C shows the results obtained for SW1736 cells.

FIGS. 68A to 68C. Graphs showing the results of analysis of the abilityof 10D1F.FcA alone, or in combination with vemurafenib, to inhibitproliferation of BRAF^(V600E)=mutant thyroid cancer cell lines in vitro.68A shows the results obtained for BHT101 cells, 68B shows the resultsobtained for SW1736 cells, and 68C shows the results obtained for BCPAPcells.

FIGS. 69A to 69C. Tables showing representative hematological profilesof BALB/c mice following administration of 10 mg/kg, 25 mg/kg, 100 mg/kgor 250 mg/kg of 10D1F.FcA or an equal volume of PBS. 69A shows resultsof analysis of the red blood cell compartment, 69B shows results ofanalysis of the white blood cell compartment, and 69C shows results ofanalysis of correlates of liver, kidney and pancreas function, andlevels of electrolytes. RBC=red blood cell, MVC=mean corpuscular volume,MCH=mean corpuscular haemoglobin, MCHC=mean corpuscular haemoglobinconcentration, WBC=white blood cell, ALT=alanine aminotransferase,ALP=alkaline phosphatase, CREA=creatinine, BUN=blood urea nitrogen,GLU=glucagon, AMY=amylase, NA=sodium, K=potassium, P=phosphorus andCA=calcium.

FIGS. 70A to 70C. Tables showing representative hematological profilesof SD rats at the indicated time points, following administration of 250mg/kg 10D1F.FcA or an equal volume of PBS. 70A shows results of analysisof the red blood cell compartment, 70B shows results of analysis of thewhite blood cell compartment, and 70C shows results of analysis ofcorrelates of liver, kidney and pancreas function, and levels ofelectrolytes. RBC=red blood cell, MVC=mean corpuscular volume, MCH=meancorpuscular haemoglobin, MCHC=mean corpuscular haemoglobinconcentration, WBC=white blood cell, ALT=alanine aminotransferase,ALP=alkaline phosphatase, CREA=creatinine, BUN=blood urea nitrogen,GLU=glucagon, AMY=amylase, NA=sodium, K=potassium, P=phosphorus andCA=calcium.

FIG. 71 . Images showing the results of analysis of the effect of10D1F.FcA treatment on HER3-mediated signalling in vivo in cells of FaDuor OvCar3 cell-derived tumors, as determined by phospho-western blot.

FIG. 72 . Box blot showing the results of analysis of internalisation ofdifferent anti-ErbB antibodies by the indicated cell lines.

FIGS. 73A and 73B. Histograms and tables showing the results of analysisof internalisation of 10D1F.FcA or trastuzumab by the indicated celllines at different time points, as determined by flow cytometry. 73Bshows median fluorescence intensity and percentages of PE-positive cellsdetermined from the histograms shown in 73A.

FIG. 74 . Graph showing the results of analysis of tumour volume overtime in a N87 cell-line derived mouse model of gastric cancer afterbiweekly treatment with the indicated concentrations of the indicatedanti-ErbB antibodies, for six weeks (n=6). Antibody administration isindicated by triangles along x-axis.

FIGS. 75A and 75B, images showing immunohistochemical staining ofmalignant and normal human tissues using 10D1F.FcA. 75A and 75B showsstaining of different tissues.

FIG. 76 . Images showing immunohistochemical staining of A549 tumorxenograft cryosections by 10D1F or a rabbit polyclonal anti-HER3antibody, at the indicated magnifications. Secondary-only controlstainings are shown.

FIGS. 77A and 77B. Bar charts showing the results of analysis of theability of the indicated anti-ErbB antibodies to inhibit in vitroproliferation of the indicated cancer cell lines at the serumconcentrations the antibodies reach at C_(max) following IPadministration to mice at 25 mg/kg. 77A and 77B show results obtainedusing different cell lines.

EXAMPLES

In the following Examples, the inventors describe the generation ofnovel anti-HER3 antibody clones targeted to specific regions of interestin the HER3 molecule, and the biophysical and functionalcharacterisation and therapeutic evaluation of these antigen-bindingmolecules.

Example 1 HER3 Target Design and Anti-HER3 Antibody Hybridoma Production

The inventors selected two regions in the extracellular region of humanHER3 (SEQ ID NO:9) for raising HER3-binding monoclonal antibodies.

1.1 Hybridoma Production

Approximately 6 week old female BALE/c mice were obtained from InVivos(Singapore). Animals were housed under specific pathogen-free conditionsand were treated in compliance with the Institutional Animal Care andUse Committee (IACUC) guidelines.

For hybridoma production, mice were immunized with proprietary mixturesof antigenic peptide, recombinant target protein or cells expressing thetarget protein.

Prior to harvesting the spleen for fusion, mice were either boosted withantigen mixture for three consecutive days or only for a single day. 24h after the final boost total splenocytes were isolated and fused withthe myeloma cell line P3X63.Ag8.653 (ATCC, USA), with PEG usingClonaCell-HY Hybridoma Cloning Kit, in accordance with themanufacturer's instructions (Stemcell Technologies, Canada).

Fused cells were cultured in ClonaCell-HY Medium C (StemcellTechnologies, Canada) overnight at 37° C. in a 5% CO₂ incubator. Thenext day, fused cells were centrifuged and resuspended in 10 ml ofClonaCell-HY Medium C and then gently mixed with 90 ml of semisolidmethylcellulose-based ClonaCell-HY Medium C (StemCell Technologies,Canada) containing HAT components, which combines the hybridomaselection and cloning into one step.

The fused cells were then plated into 96 well plates and allowed to growat 37° C. in a 5% CO₂ incubator. After 7-10 days, single hybridomaclones were isolated and antibody producing hybridomas were selected byscreening the supernatants by Enzyme-linked immunosorbent assay (ELISA)and Fluorescence-activated cell sorting (FACS).

1.2 Antibody Variable Region Amplification and Sequencing

Total RNA was extracted from hybridoma cells using TRIzol reagent (LifeTechnologies, Inc., USA) using manufacturer's protocol. Double-strandedcDNA was synthesized using SMARTer RACE 5′/3′ Kit (Clontech™, USA) inaccordance with the manufacturer's instructions. Briefly, 1 μg total RNAwas used to generate full-length cDNA using 5′-RACE CDS primer (providedin the kit), and the 5′ adaptor (SMARTer II A primer) was thenincorporated into each cDNA according to manufacturers instructions.cDNA synthesis reactions contained, 5× First-Strand Buffer, DTT (20 mM),dNTP Mix (10 mM), RNase Inhibitor (40 U/μl) and SMARTScribe ReverseTranscriptase (100 U/μl).

The race-ready cDNAs were amplified using SegAmp DNA Polymerase(Clontech™, USA). Amplification reactions contained SeqAmp DNAPolymerase, 2× Seq AMP buffer, 5′ universal primer provided in the 5′SMARTer Race kit, that is complement to the adaptor sequence, and 3′primers that anneal to respective heavy chain or light chain constantregion primer. The 5′ constant region were designed based on previouslyreported primer mix either by Krebber et al. J. Immunol. Methods 1997;201: 35-55, Wang et al. Journal of Immunological Methods 2000, 233;167-177 or Tiller et al. Journal of Immunological Methods 2009;350:183-193. The following thermal protocol was used: pre-denature cycleat 94° C. for 1 min; 35 cycles of 94° C., 30 s, 55° C., 30 s and 72° C.,45 s; final extension at 72° C. for 3 min.

The resulting VH and VL PCR products, approximately 550 bp, were clonedinto pJET1.2/blunt vector using CloneJET PCR Cloning Kit (ThermoScientific, USA) and used to transform highly competent E. coli DH5α.From the resulting transformants, plasmid DNA was prepared usingMiniprep Kb (Eugene, Germany) and sequenced. DNA sequencing was carriedout by AITbiotech. These sequencing data were analyzed using theinternational IMGT (ImMunoGeneTics) information system (LeFranc et al.,Nucleic Acids Res. (2015) 43 (Database issue):D413-22) to characterizethe individual CDRs and framework sequences. The signal peptide at 5′end of the VH and VL was identified by SignalP (v 4.1; Nielsen, inKihara, D (ed): Protein Function Prediction (Methods in MolecularBiology vol. 1611) 59-73, Springer 2017).

Four monoclonal anti-HER3 antibody clones were selected for furtherdevelopment; 10D1, 10A6, 4-35-B2, and 4-35-B4.

Humanised versions of 10D1 were designed in silico by grafting ofcomplementarity determining regions (CDRs) into VH and VL comprisinghuman antibody framework regions, and were further optimized for antigenbinding by yeast display method.

For yeast display, humanized sequences were converted into single-chainfragment variable (scFv) format by polymerase chain reaction (PCR) andused as templates to generate mutant libraries by random mutagenesis.Mutant PCR libraries were then electroporated into yeast together withlinearized pCTcon2 vector to generate yeast libraries. The librarieswere stained with human HER3 antigen and sorted for top binders. After4-5 rounds of sorting, individual yeast clones were sequenced toidentify unique antibody sequences.

Antibody done VH/VL sequence 10A6 VH = SEQ ID NO: 157 VL = SEQ ID NO:164 10D1 VH = SEQ ID NO: 24 VL = SEQ ID NO: 74 10D1_c75 VH = SEQ ID NO:25 VL = SEQ ID NO: 75 10D1_c76 VH = SEQ ID NO: 26 VL = SEQ ID NO: 7610D1_c77 VH = SEQ ID NO: 27 VL = SEQ ID NO: 77 10D1_c78v1 VH = SEQ IDNO: 28 VL = SEQ ID NO: 78 10D1_c78v2 VH = SEQ ID NO: 29 VL = SEQ ID NO:78 10D1_c11B VH = SEQ ID NO: 30 VL = SEQ ID NO: 78 10D1_c85v1 VH = SEQID NO: 31 VL = SEQ ID NO: 79 10D1_c85v2 VH = SEQ ID NO: 32 VL = SEQ IDNO: 79 10D1_c85o1 VH = SEQ ID NO: 33 VL = SEQ ID NO: 80 10D1_c85o2 VH =SEQ ID NO: 34 VL = SEQ ID NO: 81 10D1_c87 VH = SEQ ID NO: 35 VL = SEQ IDNO: 82 10D1_c89 VH = SEQ ID NO: 36 VL = SEQ ID NO: 83 10D1_c90 VH = SEQID NO: 37 VL = SEQ ID NO: 84 10D1_c91 VH = SEQ ID NO: 38 VL = SEQ ID NO:85 10D1_c92 VH = SEQ ID NO: 39 VL = SEQ ID NO: 86 10D1_c93 VH = SEQ IDNO: 40 VL = SEQ ID NO: 87 4-35-B2 VH = SEQ ID NO: 127 VL = SEQ ID NO:135 4-35-B4 VH = SEQ ID NO: 143 VL = SEQ ID NO: 150

Example 2 Antibody Production and Purification

2.1 Cloning VH and VL Into Expression Vectors:

DNA sequences encoding the heavy and light chain variable regions of theanti-HER3 antibody clones were subcloned into the pmAbaZ_IgG1_CH andpmAbDZ_IgG1_CL (InvivoGen, USA) eukaryotic expression vectors forconstruction of human-mouse chimeric antibodies.

Alternatively, DNA sequence encoding the heavy and light chain variableregions of the anti-HER3 antibody clones were subcloned into thepFUSE-CHIg-hG1 and pFUSE2ss-CLIg-hk (InvivoGen, USA) eukaryoticexpression vectors for construction of human-mouse chimeric antibodies.Human IgG1 constant region encoded by pFUSE-CHIg-hG1 comprises thesubstitutions D356E, L358M (positions numbered according to EUnumbering) in the CH3 region relative to Human IgG1 constant region(IGHG1; UniProt:P01857-1, v1; SEQ ID NO:176). pFUSE2ss-CLIg-hk encodeshuman IgG1 light chain kappa constant region (IGCK: UniProt: P01834-1,v2).

Variable regions along with the signal peptides were amplified from thecloning vector using SeqAmp enzyme (Clontech™, USA) following themanufacturer's protocol. Forward and reverse primers having 15-20 bpoverlap with the appropriate regions within VH or VL plus 6 bp at 5′ endas restriction sites were used. The DNA insert and the vector weredigested with restriction enzyme recommended by the manufacturer toensure no frameshift was introduced and ligated into its respectiveplasmid using T4 ligase enzyme (Thermo Scientific, USA). The molar ratioof 3:1 of DNA insert to vector was used for ligation.

2.2 Expression of Antibodies in Mammalian Cells

Antibodies were expressed using either 1) Expi293 Transient ExpressionSystem Kit (Life Technologies, USA), or 2) HEK293-6E TransientExpression System (CNRC-NRC, Canada) following the manufacturer'sinstructions.

1) Expi293 Transient Expression System:

Cell Line Maintenance:

HEK293F cells (Expi293F) were obtained from Life Technologies, Inc(USA). Cells were cultured in serum-free, protein-free, chemicallydefined medium (Expi293 Expression Medium, Thermo Fisher, USA),supplemented with 50 IU/ml penicillin and 50 μg/ml streptomycine (Gibco,USA) at 37° C. in 8% CO₂ and 80% humidified incubators with shakingplatform.

Transfection:

Expi293F cells were transfected with expression plasmids usingExpiFectamine 293 Reagent kit (Gibed, USA) according to itsmanufacturer's protocol. Briefly, cells at maintenance were subjected toa media exchange to remove antibiotics by spinning down the culture,cell pellets were re-suspended in fresh media without antibiotics at 1day before transfection. On the day of transfection, 2.5×10⁶/ml ofviable cells were seeded in shaker flasks for each transfection.DNA-ExpiFectamine complexes were formed in serum-reduced medium,Opti-MEM (Gibco, USA), for 25 min at room temperature before being addedto the cells. Enhancers were added to the transfected cells at 16-18 hpost transfection. An equal amount of media was topped up to thetransfectants at day 4 post-transfection to prevent cell aggregation.Transfectants were harvested at day 7 by centrifugation at 4000×g for 15min, and filtered through 0.22 μm sterile filter units.

2) HEK293-6E Transient Expression System

Cell Line Maintenance:

HEK293-6E cells were obtained from National Research Council Canada.Cells were cultured in serum-free, protein-free, chemically definedFreestyle F17 Medium (Invitrogen, USA), supplemented with 0.1%Kolliphor-P188 and 4 mM L-Glutamine (Gibco, USA) and 25 μg/ml G-418 at37° C., in 5% CO₂ and 80% humidified incubators with shaking platform.

Transfection:

HEK293-6E cells were transfected with expression plasmids using PEIpro™(Polyplus, USA) according to its manufacturer's protocol. Briefly, cellsat maintenance were subjected to a media exchange to remove antibioticsby centrifugation, cell pellets were re-suspended with fresh mediawithout antibiotics at 1 day before transfection. On the day oftransfection, 1.5-2×10⁵ cells/ml of viable cells were seeded in shakerflasks for each transfection. DNA and PEIpro™ were mixed to a ratio of1:1 and the complexes were allowed to form in F17 medium for 5 min at RTbefore adding to the cells. 0.5% (w/v) of Tryptone N1 was fed totransfectants at 24-48 h post transfection. Transfectants were harvestedat day 6-7 by centrifugation at 4000×g for 15 min and the supernatantwas filtered through 0.22 μm sterile filter units.

Cells were transfected with vectors encoding the following combinationsof polypeptides:

Antigen- biding molecule Polypeptides Antibody  [1] 10D1 VH-CH1-CH2-CH3(SEQ ID NO: 216) + anti-HER3 clone 10D1 IgG1 10D1 VL-Cκ (SEQ ID NO: 217) [2] 10A6 VH-CH1-CH2-CH3 (SEQ ID NO: 222) + anti-HER3 clone 10A6 IgG110A6 VL-Cκ (SEQ ID NO: 223)  [3] 4-35-B2 VH-CH1-CH2-CH3 (SEQ ID NO:218) + anti-HER3 clone 4-35-B2 IgG1 4-35-B2 VL-Cκ (SEQ ID NO: 219)  [4]4-35-B4 VH-CH1-CH2-CH3 (SEQ ID NO: 220) + anti-HER3 clone 4-35-B4 IgG14-35-B4 VL-Cκ (SEQ ID NO: 221)  [5] 10D1_c75 VH-CH1-CH2-CH3 (SEQ ID NO:187) + anti-HER3 clone 10D1_c75 IgG1 10D1_c75 VL-Cκ (SEQ ID NO: 188) [6] 10D1_c76 VH-CH1-CH2-CH3 (SEQ ID NO: 189) + anti-HER3 clone 10D1_c76IgG1 10D1_c76 VL-Cκ (SEQ ID NO: 190)  [7] 10D1_c77 VH-CH1-CH2-CH3 (SEQID NO: 191) + anti-HER3 clone 10D1_c77 IgG1 10D1_c77 VL-Cκ (SEQ ID NO:192)  [8] 10D1_c78v1 VH-CH1-CH2-CH3 (SEQ ID NO: 193) + anti-HER3 clone10D1_c78v1 IgG1 10D1_c78v1 VL-Cκ (SEQ ID NO: 195)  [9] 10D1_c78v1VH-CH1-CH2-CH3 (SEQ ID NO: 194) + anti-HER3 clone 10D1_c78v2 IgG110D1_c78v2 VL-Cκ (SEQ ID NO: 195) [10] 10D1_11B VH-CH1-CH2-CH3 (SEQ IDNO: 196) + anti-HER3 clone 10D1_11B IgG1 10D1_11B VL-Cκ (SEQ ID NO: 195)[11] 1QD1_c85v1 VH-CH1-CH2-CH3 (SEQ ID NO: 197) + anti-HER3 clone10D1_c85v1 IgG1 10D1_c85v1 VL-Cκ (SEQ ID NO: 199) [12] 10D1_c85v2VH-CH1-CH2-CH3 (SEQ ID NO: 198) + anti-HER3 clone 10D1_c85v2 IgG110D1_c85v2 VL-Cκ (SEQ ID NO: 199) [13] 10D1_c85o1 VH-CH1-CH2-CH3 (SEQ IDNO: 200) + anti-HER3 clone 10D1_c85o1 IgG1 10D1_c85o1 VL-Cκ (SEQ ID NO:201) [14] 10D1_c85o2 VH-CH1-CH2-CH3 (SEQ ID NO: 202) + anti-HER3 clone10D1_c85o2 IgG1 10D1_c85o2 VL-Cκ (SEQ ID NO: 203) [15] 10D1_c87VH-CH1-CH2-CH3 (SEQ ID NO: 204) + anti-HER3 clone 10D1_c87 IgG1 10D1_c87VL-Cκ (SEQ ID NO: 205) [16] 10D1_c89 VH-CH1-CH2-CH3 (SEQ ID NO: 206) +anti-HER3 clone 10D1_c89 IgG1 10D1_c89 VL-Cκ (SEQ ID NO: 207) [17]10D1_c90 VH-CH1-CH2-CH3 (SEQ ID NO: 208) + anti-HER3 clone 10D1_c90 IgG110D1_c90 VL-Cκ (SEQ ID NO: 209) [18] 10D1_c91 VH-CH1-CH2-CH3 (SEQ ID NO:210) + anti-HER3 done 10D1_c91 IgG1 10D1_c91 VL-Cκ (SEQ ID NO: 211) [19]10D1_c92 VH-CH1-CH2-CH3 (SEQ ID NO: 212) + anti-HER3 done 10D1_c92 IgG110D1_c92 VL-Cκ (SEQ ID NO: 213) [20] 10D1_c93 VH-CH1-CH2-CH3 (SEQ ID NO:214) + anti-HER3 done 10D1_c93 IgG1 10D1_c93 VL-Cκ (SEQ ID NO: 215)

2.3 Antibody Purification

Affinity Purification, Buffer Exchange and Storage:

Antibodies secreted by the transfected cells into the culturesupernatant were purified using liquid chromatography system AKTA Start(GE Healthcare, UK). Specifically, supernatants were loaded onto HiTrapProtein G column (GE Healthcare, UK) at a binding rate of 5 ml/min,followed by washing the column with 10 column volumes of washing buffer(20 mM sodium phosphate, pH 7.0). Bound mAbs were eluted with elutionbuffer (0.1 M glycine, pH 2.7) and the eluents were fractionated tocollection tubes which contain appropriate amount of neutralizationbuffer (1 M Tris, pH 9). Neutralised elution buffer containing purifiedmAb were exchanged into PBS using 30K MWCO protein concentrators (ThermoFisher, USA) or 3.5K MWCO dialysis cassettes (Thermo Fisher, USA).Monoclonal antibodies were sterilized by passing through 0.22 μm filter,aliquoted and snap-frozen in −80° C. for storage.

2.4 Antibody-Purity Analysis

Size Exclusion Chromatography (SEC):

Antibody purity was analysed by size exclusion chromatography (SEC)using Superdex 200 10/30 GL columns (GE Healthcare, UK) in PBS runningbuffer, on a AKTA Explorer liquid chromatography system (GE Healthcare,UK). 150 μg of antibody in 500 μl PBS pH 7.2 was injected to the columnat a flow rate of 0.75 ml/min at room temperature. Proteins were elutedaccording to their molecular weights.

The result for anti-HER3 antibody clone 10D1 ([1] of Example 2.2) isshown in FIG. 12 .

The results obtained for the different 10D1 variant clones are shown inFIG. 34 .

Sodium-Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE):

Antibody purity was also analysed by SDS-PAGE under reducing andnon-reducing conditions according to standard methods. Briefly, 4%-20%TGX protein gels (Bio-Rad, USA) were used to resolve proteins using aMini-Protean Electrophoresis System (Bio-Rad, USA). For non-reducingcondition, protein samples were denatured by mixing with 2× Laemmlisample buffer (Bio-Rad, USA) and boiled at 95° C. for 5-10 min beforeloading to the gel. For reducing conditions, 2× sample buffer containing5% of β-mercaptoethanol (βME), or 40 mM DTT (dithiothreitol) was used.Electrophoresis was carded out at a constant voltage of 150V for 1 h inSDS running buffer (25 mM Tris, 192 mM glycine, 1% SDS, pH 8.3).

Western Blot:

Protein samples (30 μg) were fractionated by SDS-PAGE as described aboveand transferred to nitrocellulose membranes. Membranes were then blockedand immunoblotted with antibodies overnight at 4° C. After washing threetimes in PBS-Tween the membranes were then incubated for 1 h at roomtemperature with horseradish peroxidase (HRP)-conjugated secondaryantibodies. The results were visualized via a chemiluminescent PierceECL Substrate Western blot detection system (Thermo Scientific, USA) andexposure to autoradiography film (Kodak XAR film).

The primary antibodies used for detection were goat anti-human IgG-HRP(GenScript Cat No. A00166) and goat anti-human kappa-HRP(SouterhnBiotech Cat No. 2060-05).

The result for anti-HER3 antibody clone 10D1 ([1] of Example 2.2) isshown in FIG. 13 . 10D1 was easily expressed, purified and processed athigh concentrations.

Example 3 Biophysical Characterisation

3. 1 Analysis of Cell Surface Antigen-Binding by Flow Cytometry

Wildtype HEK293 cells (which do not express high levels of HER3) andHEK293 cells transfected with vector encoding human HER3 (i.e. HEK 293HER O/E cells) were incubated with 20 μg/ml of anti-HER3 antibody orisotype control antibody at 4° C. for 1.5 hr. The anti-HER3 antibodyclone LJM716 (described e.g. in Garner et al., Cancer Res (2013) 73:6024-6035) was included in the analysis as a positive control.

The cells were washed thrice with FACS buffer (PBS with 5 mM EDTA and0.5% BSA) and resuspended in FITC-conjugated anti-FC antibody(Invitrogen, USA) for 40 min at 2-8° C. Cells were washed again andresuspended in 200 μL of FACS flow buffer (PBS with 5 mM EDTA) for flowcytometric analysis using MACSQuant 10 (Miltenyi Biotec, Germany). Afteracquisition, all raw data were analyzed using Flowlogic software. Cellswere gated using forward and side scatter profile percentage of positivecells was determined for native and overexpressing cell populations.

The results are shown in FIGS. 1 to 4 and 30 to 32 . The anti-HER3antibodies were shown to bind to human HER3 with high specificity. 10D1and LJM716 were shown to bind to human HER3-expressing cells to asimilar extent.

3.2 ELISAs for Determining Antibody Specificity and Cross-Reactivity

ELISAs were used to determine the binding specificity of the antibodies.The antibodies were analysed for binding to human HER3 polypeptide, aswell as respective mouse, rat and monkey homologues of HER3 (SinoBiological Inc., China). The antibodies were also analysed for theirability to bind to human EGFR and human HER2 (Sino Biological Inc.,China).

ELISAs were carded out according to standard protocols. Briefly, 96-wellplates (Nunc, Denmark) were coated with 0.1 μg/ml of target polypeptidein phosphate-buffered saline (PBS) for 16 h at 4° C. After blocking for1 h with 1% BSA in Tris buffer saline (TBS) at room temperature, theanti-HER3 antibody was serially diluted with the highest concentrationbeing 10 μg/ml, and added to the plate. Post 1 h incubation at roomtemperature, plates were washed three times with TBS containing 0.05%Tween 20 (TBS-T) and were then incubated with a HRP-conjugated anti-Hisantibody (Life Technologies, Inc., USA) for 1 h at room temperature.After washing, plates were developed with colorimetric detectionsubstrate 3,3′,5,5′-tetramethylbenzidine (Turbo-TMB; Pierce, USA) for 10min. The reaction was stopped with 2M H₂SO₄, and OD was measured at 450nM.

The results of the ELISAs are shown in FIGS. 5 to 7 and FIG. 33 .

Anti-HER3 antibody clone 10D1 was found not to bind to human HER2 orhuman EGFR even at high concentrations of the antibody (FIG. 5A).Anti-HER3 antibody clone 10D1 was also found to display substantialcross-reactivity with mouse HER3, rat HER3 and cynomolgus macaque HER3(FIG. 5B).

Anti-HER3 antibody clone 4-35-B2 was found to bind to human HER2 andhuman EGFR (FIG. 6A). Anti-HER3 antibody clone 4-35-B2 also displayedsubstantial cross-reactivity with mouse HER3, rat HER3 and cynomolgusmacaque HER3 (FIG. 6B).

Anti-HER3 antibody clone 4-35-B4 was found to bind to human HER2 andhuman EGFR (FIG. 7A). Anti-HER3 antibody clone 4-35-B4 also displayedsubstantial cross-reactivity with mouse HER3, rat HER3 and cynomolgusmacaque HER3 (FIG. 7B).

All of the 10D1 variants were demonstrated to bind to human HER3 (FIGS.33A and 33B).

3.3 Global Affinity Study Using Octet QK384 System

The anti-HER3 antibody clones in IgG1 format were analysed for bindingaffinity to human HER3.

Bio-Layer Interferometry (BLE) experiments were performed using theOctet QK384 system (ForteBio). anti-Human IgG Capture (AHC) Octet sensortips (Pall ForteBio, USA) were used to anti-HER3 antibodies (25 nM). Allmeasurements were performed at 25° C. with agitation at 1000 rpm.Kinetic measurements for antigen binding were performed by loadingHis-tagged human HER3 antigens at different concentrations for 120 s,followed by a 120 s dissociation time by transferring the biosensorsinto assay buffer containing wells. Sensograms were referenced forbuffer effects and then fitted using the Octet QK364 user software (PallForteBio, USA). Kinetic responses were subjected to a global fittingusing a one site binding model to obtain values for association(K_(on)), dissociation (K_(off)) rate constants and the equilibriumdissociation constant (K_(D)). Only curves that could be reliably fittedwith the software (R²>0.90) were included in the analysis.

A representative sensorgram for the analysis of clone 10D1 is shown inFIG. 8 . Clone 10D1 was found to bind to human HER3 with an affinity ofK_(D)=9.58 nM.

The humanised/optimized 10D1 variants bind to human HER3 with very highaffinity. Representative sensorgrams are shown in FIGS. 36A to 36M.

The affinities determined for 10D1 clone variants are shown below:

Antibody clone Affinity (K_(D)) 10D1_c89 72.6 pM 10D1_c90 <1 pM 10D1_c91176 pM 10D1_11B 0.41 nM 10D1_c85o 17.3 nM 10D1_c87 <1 pM 10D1_c93 <1 pM10D1_c76 <1 pM 10D1_c77 1.93 nM 10D1_c78 <1 pM 10D1_c75 <1 pM 10D1_c857.58 nM 10D1_c85o1 18.2 nM

Clone 4-35-B2 was found to bind to human HER3 with an affinity ofK_(D)=80.9 nM (FIG. 9 ), and clone 4-35-B4 was found to bind to humanHER3 with an affinity of K_(D)=50.3 nM (FIG. 10 ).

3.4 Analysis of Thermostability by Differential Scanning Fluorimetry

Briefly, triplicate reaction mixes of antibodies at 0.2 mg/mL and SYPROOrange dye (ThermoFisher) were prepared in 25 μL of PBS, transferred towells of MicroAmp Optical 96-Well Reaction Plates (ThermoFisher), andsealed with MicroAmp Optical Adhesive Film (ThermoFisher). Meltingcurves were run in a 7500 fast Real-Time PCR system (Applied Biosystems)selecting TAMRA as reporter and ROX as passive reference. The thermalprofile included an initial step of 2 min at 25° C. and a final step of2 min at 99° C., with a ramp rate of 1.2%. The first derivative of theraw data was plotted as a function of temperature to obtain thederivative melting curves. Melting temperatures (Tm) of the antibodieswere extracted from the peaks of the derivative curves.

The first derivative of the raw data obtained for Differential ScanningFluorimetry analysis of the thermostability of antibody clone 10D1 isshown in FIG. 11 . Three different samples of the antibody wereanalysed. The Tm was determined to be 70.3° C.

The analysis was also performed for the 10D1 variant clones and LJM716.The first derivative of the raw data and the determined Tms are shown inFIGS. 35A to 35C.

3.5 Analysis of Anti-HER3 Antibody 10D1 Epitope

Anti-HER3 antibody 10D1 was analysed to determine whether it competeswith anti-HER3 antibodies MM-121 and/or LJM-716 for binding to HER3. Theepitope for MM-121 has been mapped in domain 1 of HER3; it blocks theNRG ligand binding site. The epitope for LJM-716 has been mapped toconformational epitope distributed across domains II and IV, and itlocks HER3 in an inactive conformation.

Bio-Layer Interferometry (BLI) experiments were performed using theOctet QK384 system (ForteBio). anti-Penta-HIS (HIS1K) coated biosensortips (ForteBio, USA) were used to capture His-tagged human HER3 (75 nM;300 s). Binding by saturating antibody (400 nm; 600 s) was detected,followed by a dissociation step (120 s), followed by detection ofbinding with competing antibody (300 nM; 300 s), followed by adissociation step (120 s). The variable region of MM-121 antibody wascloned in the PDZ vector having human IgG2 and IgKappa Fc backbone. Thevariable region of LJM-716 antibody was cloned in the PDZ vector havinghuman IgG1 and IgKappa Fc backbone.

The results of the analysis are shown in FIGS. 14A and 14B. Anti-HER3antibody was found not to compete with MM-121 and/or LJM-718 for bindingto HER3.

10D1 was found to bind a distinct and topologically distant epitope ofHER3 than MM-121 and/or LJM-716.

The epitope for 10D1 was mapped using overlapping 15-mer amino acids tocover the entire HER3 extracellular domain. Each unique 15-mer waselongated by a GS linker at C and N-terminals, conjugated to a uniquewell in 384 well plates, and the plates were incubated with 0.1, 1, 10and 100 ug/ml of 10D1 antibody for 16 hrs at 4° C. The plates werewashed and then incubated for 1 hr at 20° C. with POD-conjugated goatanti-human IgG. Finally POD substrate solution was added to the wellsfor 20 min. before binding was assessed by measurement ofchemiluminescence at 425 nm using a LI-COR Odyssey Imaging System, andquantification and analysis was performed using the PepSlide Analyzersoftware package. The experiment was performed in duplicate.

The 10D1 epitope was found not to be directly located at a β-hairpinstructure of the HER3 dimerisation arm located at domain II, but insteadat a dimerisation interface N-terminal to the β-hairpin.

The site of HER3 to which 10D1 and 10D1-derived clones was determined tobind corresponds to positions 218 to 235 of the amino acid sequence ofhuman HER3 (as shown e.g. in SEQ ID NO:1); the amino acid sequence forthis region of HER3 is shown in SEQ ID NO:229. Within this region, twoconsensus binding site motifs were identified, and are shown in SEQ IDNOs:230 and 231.

Binding to this location of HER3 acts to impede HER familyheterodimerisation and consequent downstream signalling pathways (seeExample 4). Binding is ligand (NRG) independent. The 10D1 binding siteis solvent accessible in both the open and closed HER3 conformations, isnot conserved between HER3 and other HER family members, and is 100%conserved between human, mouse, rat and monkey HER3 orthologs.

Example 4 Functional Characterisation

4.1 Inhibition of Dimerisation of HER2 and HER3

The anti-HER3 antibodies were analysed for their ability to inhibitheterodimerisation of HER3 and HER2.

Briefly, 96-well plates (Nunc, Denmark) were coated with 0.1 μg/mlHis-tagged HER2 protein in PBS for 16 h at 4° C. After blocking for 1 hwith 1% BSA in PBS at room temperature, recombinant biotinylated humanHER3 protein was added in the presence of different concentrations ofanti-HER3 antibody clone 10D1, and plates were incubated for 1 h at roomtemperature. Plates were subsequently washed three times, and thenincubated with HRP-conjugated secondary antibody for 1 h at roomtemperature. After washing, plates were developed with colorimetricdetection substrate 3,3′,5,5′-tetramethylbenzidine (Turbo-TMB; Pierce,USA) for 10 min. The reaction was stopped with 2M H₂SO₄, and OD wasmeasured at 450 nM.

The results are shown in FIG. 15 . Anti-HER3 antibody clone 10D1 wasfound to inhibit interaction between HER2 and HER3 in a dose-dependentfashion.

In further experiments, inhibition of HER2:HER3 dimerisation wasanalysed

In further experiments, inhibition of HER2:HER3 dimerisation wasevaluated using the PathHunter Pertuzumab Bioassay Kit (DiscoverX)according to the manufacturer's instructions.

Briefly, HER2 and HER3 overexpressing U2OS cells were thawed using 1 mlof pre-warmed CP5 media and 5,000 cells were seeded per well andcultured at 37° C. in 5% CO₂ atmosphere for 4 hr. Cells were thentreated with an 8-point serial dilution of 10D1F.FcA or Pertuzumab,starting from 25 μg/ml.

After 4 hr incubation, 30 ng/ml of heregulin-β2 was added to each welland the cells were incubated for a further 16 hr. 10 μL of PathHunterbioassay detection reagent 1 was added to wells, and incubated for 15min at room temperature in the dark. This was followed by addition of 40μL PathHunter bioassay detection reagent 2, and incubation for 60 min atroom temperature in the dark. Plates were then read using Synergy4Biotek with 1 second delay.

The results are shown in FIG. 65 . 10D1F.FcA was found inhibit HER2:HER3dimerisation with greater efficiency than pertuzumab, as reflected byits lower IC50.

4.2 identification of Cancer Cell Lines for Analysis

The inventors characterised expression of EGFR protein family members bycancer cell lines to identify appropriate cells to investigateinhibition of HER3.

FIG. 16A shows mRNA expression data EGFR family members and ligands byN87, SNU16, HT29, FaDu, A549, HCC95, OvCAR8 and AHCN cells according tothe Cancer Cell Line Encyclopaedia (CCLE; Barretina et al., Nature(2012) 483: 603-607 and The Cancer Cell Line Encyclopedia Consortium &The Genomics of Drug Sensitivity in Cancer Consortium, Nature (2015)528: 84-87). FIG. 16A also shows protein expression data for EGFR, HER2and HER3 as determined by FlowLogic.

Cell lines used in the experiments were purchased from ATCC and culturedas recommended. Briefly, cell lines maintained in the indicated cellculture medium, supplemented with 10% FBS and 1% Pen/Strep. Cells werecultured at 37° C., in 5% CO₂ incubators. Cultured cells were plated atthe appropriate seeding density in a 96 well plate: HT29, HCC95, FADUand OvCar8 cells were seeded at 2000 cells/well, NCI-N87 cells wereseeded at 5000 cells/well, SNU-16, ACHN and cells were seeded at 1500cells/well, and A549 cells were seeded at 1200 cells/well.

FIG. 16B shows surface expression of EGFR, HER2 and HER3 as determinedby flow cytometry. Briefly, 500,000 cells were stained in stainingbuffer containing 0.5% BSA and 2 mM EDTA with primary antibodies (20μg/ml) for 1.5 h at 4° C. The secondary Antibody used was anti-humanAlexafluor488 at 10 μg/ml for 20 min at 4° C.

4.3 Inhibition of HER3-Mediated Signalling

Anti-HER3 antibody 10D1 was analysed for its ability to inhibit HER-3mediated signalling in vitro.

Briefly, N87 and FaDu cells were seeded in wells of a 6 well plate with10% serum at 37° C., 5% CO₂. After 16 hrs, cells were starved by cultureovernight in 1% FBS cell culture medium (to reduce signalling elicitedby growth factors in the serum). On the following day cells were treatedwith 50 μg/ml anti-HER3 antibody 10D1 for 4 hrs, followed by 15 minstimulation with NRG (100 ng/ml). Proteins were then extracted,quantified using standard Bradford protein assay, fractionated bySDS-PAGE, and transferred to nitrocellulose membranes. The membraneswere then blocked and immunoblotted with the following antibodiesovernight at 4° C. anti-pHER3, anti-pAKT, pan anti-HER3, pan anti-AKTand anti-beta-actin. The blots were visualized via Bio-Rad ClarityWestern ECL substrate, and bands were quantified using densiometricanalysis; data were normalized to beta actin controls.

The results are shown in FIG. 17 . Anti-HER3 antibody 10D1 was found toinhibit HER3 phosphorylation and downstream signalling.

In further experiments the inventors investigated the intracellularsignalling pathways affected by anti-HER3 antibody-mediated inhibitionof HER3.

FaDu cells were seeded in wells of a 6 well plate with 10% serum at 37°C. 5% CO₂. After 16 hrs, cells were starved by culture overnight in 1%FBS cell culture medium. On the following day cells were treated with 50μg/ml anti-HER3 antibody 10D1 for 4 hrs, followed by 15 min stimulationwith NRG (100 ng/ml). Proteins were then extracted, quantified usingstandard Bradford protein assay, and incubated overnight withpre-blocked Phosphoprotein Antibody Array membrane (Ray Biotech) at 4°C. The membrane was then washed with washing buffer and incubated withdetection antibody cocktail for 2 hrs at room temperature, followed bywashing and incubation with HRP-Conjugated anti-IgG. After 2 hrs themembrane was washed and probed using the kit detection buffer. Imageswere captured with Syngene Gbox imaging system, the intensity of eachdot/phosphoprotein was measured and percent inhibition was calculated bycomparison with intensity measured for cells treated in the same way inthe absence of the antibody.

The results are shown in FIG. 18 . Anti-HER3 antibody 10D1 was found toinhibit PI3K/AKT/mTOR and MAPK signalling.

In further experiments the inventors investigated the effect oftreatment with anti-HER3 antibody 10D1 on proliferation ofHER3-expressing cells.

Briefly, N87 and FaDu cells were treated with serially dilutedconcentrations of anti-HER3 antibody 10D1, starting from 100 μg/ml witha 9-point half log dilution. Cell proliferation was measuring using theCCK-8 proliferation assay (Dojindo, Japan) after a period of 5 days, inaccordance with the manufacturer's instructions. Briefly 1× CCK-8solution was added to each well followed by incubation for 2 h at 37° C.The OD was then measured at 450 nm.

FIGS. 19A and 19B shows the percent cell confluence relative tountreated control cells (data points are averages of three replicates).

Anti-HER3 antibody 10D1 displayed dose-dependent inhibition of cellproliferation by N87 and FaDu cells.

Example 5 Analysis In Vivo

5.1 Pharmacokinetic Analysis

Female NCr nude mice approximately 6-8 weeks old were housed underspecific pathogen-free conditions and treated in compliance with theInstitutional Animal Care and Use Committee (IACUC) guidelines.

500 μg anti-HER3 antibody was administered and blood was obtained from 3mice by cardiac puncture at baseline (−2 hr), 0.5 hr, 6 hr, 24 hr, 96hr, 168 hr and 336 hr after administration. Antibody in the serum wasquantified by ELISA.

The results are shown in FIG. 50 . Anti-HER3 antibody clone 10D1 wasfound to have a half-life of 16.3 days in NCr nude mice.

5.2 Safety Immunotoxicity

Anti-HER3 antibody clone 10D1 was analysed in silico for safety andimmunogenicity using IMGT DomainGapAlign (Ehrenmann et al., NucleicAcids Res., 38, D301-307 (2010)) and IEDB deimmunization (Dhanda et al.,Immunology. (2018) 153(1):118-132) tools.

Anti-HER3 antibody clone 10D1 had numbers of potential immunogenicpeptides few enough to be considered safe, and did not possess any otherproperties that could cause potential developability issues.

The Table of FIG. 37 provides an overview of the properties of the 10D1variant clones relevant to safety and developability.

Mice treated with anti-HER3 antibodies in the experiments described inExample 5.3 were monitored for changes in weight and gross necroscopy.No differences were detected in these mice as compared to mice treatedwith vehicle only.

Hemotoxicity was investigated in an experiment in which 6-8 week oldfemale BALB/c mice (20-25 g) were injected intraperitoneally with asingle dose of 1000 μg anti-HER3 10D1 antibody or an equal volume ofPBS. Blood samples were obtained at 96 hours post injection and analysedfor numbers of different types of white blood cells by flow cytometryand electrolyte indices for NA⁺, K⁺, and Cl⁻.

FIGS. 51A and 51B show that the numbers of the different cell types andelectrolyte indices were found to be within the Charles River referencerange (3 mice) and did not differ significantly from the PBS-treatedgroup (3 mice). Left bars represent vehicle, right bars represent 10D1treatment, end points of the Charles River reference range indicatedwith dotted lines. No differences in clinical signs, gross necroscopy orweight were detected between the different groups.

Mice were also analysed for correlates hepatotoxicity, nephrotoxicityand pancreatic toxicity at 96 hours post injection. The levels ofalanine aminotransferase (ALT), aspartate transaminase (AST), blood ureanitrogen (BUN), creatinine (CREA), alkaline phosphatase (ALP), glucose(GLU), calcium (CAL), total bilirubin (BIL), total protein (TPR) andalbumin (ALB) detected following administration of a single dose of 1000μg anti-HER3 antibody were found to be within the Charles Riverreference range and do not differ significantly from the levels of thesemarkers in the PBS-treated group. These are shown in FIGS. 51C to 51F.Left bars represent vehicle, right bars represent 10D1 treatment, endpoints of the Charles River reference range indicated with dotted lines.10D1 treatment has no effect on the kidney, liver or pancreatic indicesand thus does not affect normal kidney, liver or pancreatic functions.

5.3 Analysis of Efficacy to Treat Cancer In Vivo

Female NCr nude mice approximately 6-8 weeks old were purchased fromInVivos (Singapore). Animals were housed under specific pathogen-freeconditions and were treated in compliance with the Institutional AnimalCare and Use Committee (IACUC) guidelines.

Cell lines used included N87 cells (gastric cancer), FaDu cells (headand neck cancer), OvCAR8 cells (ovarian cancer), SNU16 cells (gastriccancer), HT29 cells (colorectal cancer), A549 cells (lung cancer), HCC95cells (lung cancer) and AHCN cells (kidney cancer).

Tumor volumes were measured 3 times a week using a digital caliper andcalculated using the formula [L×W2/2]. Study End point was considered tohave been reaches once the tumors of the control arm measured >1.5 cm inlength.

5.3.1 N87 Model

FIG. 20 shows the results obtained in an experiment wherein theanti-cancer effect of anti-HER3 antibody 10D1 ([1] of Example 2.2) wasinvestigated in a N87 cell-line derived mouse gastric carcinoma model.The model was established by subcutaneous injection of 1×10⁶ N87 cellsinto the right flank (n=6 mice per treatment group).

10D1 was administered IP, biweekly at 500 μg per dose (for a total of 10doses); a control treatment group received an equal volume of PBS.

Anti-HER3 antibody clone 10D1 was found to be highly potent in thismodel, and capable of inhibiting tumor growth by 76%.

FIG. 21 shows the results obtained in a similar experiment in whereinanti-HER3 antibody clone 4-35-B4 was administered weekly IP at a dose of11 mg/kg (total of 4 doses). Anti-HER3 antibody clone 4-35-B4 wassimilarly found to be highly potent in this model, and capable ofinhibiting tumor growth by 60%.

5.3.2 SNU16 Model

FIG. 22 shows the results obtained in an experiment wherein theanti-cancer effect of anti-HER3 antibody 10D1 ([1] of Example 2.2) wasinvestigated in a SNU16 cell-line derived mouse gastric carcinoma model.The model was established by subcutaneous injection of 1×10⁶ SNU16 cellsinto the right flank (n=6 mice per treatment group).

10D1 was administered IP, biweekly at 500 μg per dose (for a total of 9doses); a control treatment group received an equal volume of PBS.

Anti-HER3 antibody clone 10D1 was found to be highly potent in thismodel, and capable of inhibiting tumor growth by 68%.

5.3.3 FaDu Model

FIG. 23 shows the results obtained in an experiment wherein theanti-cancer effect of anti-HER3 antibody 10D1 ([1] of Example 2.2) wasinvestigated in a FaDu cell-line derived mouse model of head and necksquamous cell carcinoma. The model was established by subcutaneousinjection of 1×10⁶ FaDu cells into the right flanks of female NPG mice(NOD scid gamma phenotype; n=6 mice per treatment group).

10D1 was administered IP, weekly at 500 μg per dose (for a total of 4doses). Control treatment groups received an equal volume of PBS, or thesame dose of an isotype control antibody.

Anti-HER3 antibody clone 10D1 was found to be highly potent in thismodel, and capable of inhibiting tumor growth by 85%.

FIG. 24 shows the results obtained in an experiment wherein theanti-cancer effect of anti-HER3 antibody 10D1 ([1] of Example 2.2) wasinvestigated in a FaDu cell-line derived mouse model of head and necksquamous cell carcinoma. The model was established by subcutaneousinjection of 1×10⁶ FaDu cells into the right flanks of female NCr nudemice (n=6 mice per treatment group).

10D1 was administered IP, biweekly at 500 μg per dose (for a total of 8doses); a control treatment group received an equal volume of PBS.

Anti-HER3 antibody clone 10D1 was found to be highly potent in thismodel, and capable of inhibiting tumor growth by 86%.

5.3.4 OvCAR8 Model

FIG. 25 shows the results obtained in an experiment wherein theanti-cancer effect of anti-HER3 antibody 10D1 ([1] of Example 2.2) wasinvestigated in an OvCAR8 cell-line derived mouse model of ovariancarcinoma. The model was established by subcutaneous injection of 1×10⁶OvCAR8 cells into the right flanks of female NCr nude mice (n=6 mice pertreatment group).

10D1 was administered IP, biweekly at 500 μg per dose (for a total of 9doses); a control treatment group received an equal volume of PBS.

Anti-HER3 antibody clone 10D1 was found to be highly potent in thismodel, and capable of inhibiting tumor growth by ˜74%.

5.3.5 HCC-95 Model

FIG. 26 shows the results obtained in an experiment wherein theanti-cancer effect of anti-HER3 antibody 10D1 ([1] of Example 2.2) wasinvestigated in a HCC-95 cell-line derived mouse model of squamous celllung carcinoma. The model was established by subcutaneous injection of1×10⁶ HCC-95 cells into the right flanks of female NCr nude mice (n=6mice per treatment group).

10D1 was administered IP, biweekly at 500 μg per dose (for a total of 4doses); a control treatment group received an equal volume of PBS.

Anti-HER3 antibody clone 10D1 was found to be highly potent in thismodel, and capable of inhibiting tumor growth by 90%.

5.3.6 A549 Model

FIG. 27 shows the results obtained in an experiment wherein theanti-cancer effect of anti-HER3 antibody 10D1 ([1] of Example 2.2) wasinvestigated in an A549 cell-line derived mouse model of lungadenocarcinoma. The model was established by subcutaneous injection of1×10⁶ A549 cells into the right flanks of female NCr nude mice (n=6 miceper treatment group).

10D1 was administered IP, biweekly at 500 μg per dose (for a total of 10doses); a control treatment group received an equal volume of PBS.

Anti-HER3 antibody clone 10D1 was found to be highly potent in thismodel, and capable of inhibiting tumor growth by 91%.

FIG. 28 shows the results obtained in a similar experiment in whereinthe anti-cancer effect of anti-HER3 antibody clone 4-35-B2 wasinvestigated in an A549 cell-line derived model established by injectionof 1×10⁶ A549 cells into the right flanks of female NPG mice (NOD scidgamma phenotype). Anti-HER3 antibody clone 4-35-B2 was administered IPweekly at a dose of 500 μg per dose (total of 4 doses). A controltreatment group received an equal volume of PBS (6 mice per treatmentgroup).

Anti-HER3 antibody clone 4-35-B2 was similarly found to be highly potentin this model, and capable of inhibiting tumor growth by 63%.

5.3.6 ACHN Model

FIG. 29 shows the results obtained in an experiment wherein theanti-cancer effect of anti-HER3 antibody 10D1 ([1] of Example 2.2) wasinvestigated in an ACHN cell-line derived mouse model of renal cellcarcinoma. The model was established by subcutaneous injection of 1×10⁶ACHN cells into the right flanks of female NCr nude mice (n=6 mice pertreatment group).

10D1 was administered IP, biweekly at 500 μg per dose (for a total of 7doses); a control treatment group received an equal volume of PBS.

Anti-HER3 antibody clone 10D1 was found to be highly potent in thismodel, and capable of inhibiting tumor growth by 61%.

5.4 Treatment of Gastric Carcinoma

First in Human

Patients with HER2+ advanced gastric cancer who have failed or cannotreceive trastuzumab are treated by intravenous injection of anti-HER3antibody selected from: 10D1, 10D1_c75, 10D1_c76, 10D1_c77, 10D1_c78v1,10D1_c78v2, 10D1_11B, 10D1_c85v1, 10D1_c85v2, 10D1_c85o1, 10D1_c85o2,10D1_c87, 10D1_c89, 10D1_c90, 10D1_c91, 10D1_c92 and 10D1_c93, at a dosecalculated in accordance with safety-adjusted ‘Minimal AnticipatedBiological Effect Level’ (MABEL) approach. Patients are monitored for 28days post-administration.

The patients are then evaluated according to the Common TerminologyCriteria for Adverse Events (CTCAE), to determine the safety andtolerability of the treatment, and to determine the pharmacokinetics ofthe molecules.

Treatment with the anti-HER3 antibodies is found to be safe andtolerable.

Dose Escalation—Monotherapy

12-48 patients with HER2+ advanced gastric cancer who have failed orcannot receive trastuzumab are treated by intravenous injection ofanti-HER3 antibody selected from: 10D1, 10D1_c75, 10D1_c76, 10D1_c77,10D1_c78v1, 10D1_c78v2, 10D1_11B, 10D1_c85v1, 10D1_c85v2, 10D1_c85o1,10D1_c85o2, 10D1_c87, 10D1_c89, 10D1_c90, 10D1_c91, 10D1_c92 and10D1_c93 (e.g. 10D1_c89, 10D1_c90 or 10D1_c91; e.g. 10D1_c89), inaccordance with a 3+3 model based escalation with overdose control(EWOC) dose escalation.

The patients are then evaluated according to the Common TerminologyCriteria for Adverse Events (CTCAE) to determine the safety andtolerability of the treatment, and the pharmacokinetics of the moleculesand efficacy of the treatment is evaluated. The maximum tolerated dose(MTD) and maximum administered dose (MAD) are also determined.

Dose Escalation—Combination Therapy

9-18 patients with HER2+ advanced gastric cancer who have failed ortrastuzumab are treated by intravenous injection of anti-HER3 antibodyselected from: 10D1, 10D1_c75, 10D1_c76, 10D1_c77, 10D1_c78v1,10D1_c78v2, 10D1_11B, 10D1_c85v1, 10D1_c85v2, 10D1_c85o1, 10D1_c85o2,10D1_c87, 10D1_c89, 10D1_c90, 10D1_c91, 10D1_c92 and 10D1_c93 (e.g.10D1_c89, 10D1_c90 or 10D1_c91; e.g. 10D1_c89) in combination withtrastuzumab, in accordance with a 3+3 model based escalation withanti-PD-L1 antibody (3 mg/kg).

The patients are then evaluated according to the Common TerminologyCriteria for Adverse Events (CTCAE) to determine the safety andtolerability of the treatment, and the pharmacokinetics of the moleculesand efficacy of the treatment is evaluated.

Dose Expansion

Patients with HER2+ advanced gastric cancer who have recently failedtrastuzumab, and whose tumours have been well-characterised geneticallyand histologically are treated with anti-HER3 antibody selected from:10D1, 10D1_c75, 10D1_c76, 10D1_c77, 10D1_c78v1, 10D1_c78v2, 10D1_11B,10D1_c85v1, 10D1_c85v2, 10D1_c85o1, 10D1_c85o2, 10D1_c87, 10D1_c89,10D1_c90, 10D1_c91, 10D1_c92, 10D1_c93 (e.g. 10D1_c89, 10D1_c90 or10D1_c91; e.g. 10D1_c89) in combination with trastuzumab, cisplatin, andeither 5-FU or capecitabine

The anti-HER3 antibodies are found to be safe and tolerable, to be ableto reduce the number/proportion of cancer cells, reduce tumor cellmarker expression, increase progression-free survival and increaseoverall survival.

Example 6 Affinity Matured and Humanised Clones

Humanization of the variable regions of the parental mouse antibody10D1P was done by CDR grafting. Human framework sequences for graftingwere identified by blasting the parental amino acid sequence against thehuman V domain database and the genes with highest identity to theparental sequence were selected. Upon grafting the mouse CDRs into theselected human frameworks, residues in canonical positions of theframework were back mutated to the parental mouse sequence to preserveantigen binding. A total of 9 humanized variants of 10D1P were designed.

Affinity against human HER3 was increased by two rounds of affinitymaturation using yeast display. In the first round, a mixed library ofthe 9 designed variants was constructed by random mutagenesis andscreened by flow cytometry using biotinylated antigen. In the secondround, one heavy chain and one light chain clones isolated in the firstround were used as template to generate and screen a second library. Atotal of 10 humanized and affinity matured clones were isolated.

Potential liabilities (immunogenicity, glycosylation sites, exposedreactive residues, aggregation potential) in the variable regions of thedesigned and isolated humanized variants of 10D1P was assessed using insilico prediction tools. The sequences were deimmunised using EDBdeimmunisation tool. The final sequence of 10D1F was selected among theoptimized variants based on its developability characteristics as wellas in vitro physicochemical and functional properties.

Clone 10D1F comprises VH of SEQ ID NO:36 and VL of SEQ ID NO:83. 10D1Fdisplays 89.9% homology with human heavy chain and 35.3% homology withhuman light chain.

The antigen-binding molecule comprising 10D1F variable regions and humanIgG1 constant regions, and which is comprised of the polypeptides of SEQID NOs: 206 and 207, is designated 10D1F.FcA (also sometimes referred toherein as “10D1F.A” or “anti-HER3 clone 10D1_c89 IgG1”—see e.g. [16] ofExample 2.2).

Example 7 Fc Engineering

10D1 and 10D1 variants were engineered to comprise mutations in CH2and/or CH3 regions to increase the potency of the antibodies, e.g.optimise Fc effector function, enhance antibody-dependent cellularcytotoxicity (ADCC) and/or antibody dependent cellular phagocytosis(ADCP), and improve half-life.

The Fc regions of clones 10D1 and 10D1F.FcA were modified to includemodifications ‘GASDALIE’ (G236A, S239D, A330L, I332E) and ‘LCKC’ (L242C,K334C) in the CH2 region. The GASDALIE substitutions were found toincrease affinity for the FcγRIIa (GA) and FcγRIIIa (SDALIE) receptorsand enhance ADCP and NK-mediated ADCC (see Example 3.8), whilstdecreasing affinity for C1q (AL) and reducing CDC. The LCKCsubstitutions were found to increase thermal stability of the Fc regionby creating a new intramolecular disulphide bridge.

The modified version of 10D1F.FcA heavy chain polypeptide comprising theGASDALIE and LCKC mutations is shown in SEQ ID NO:225. Theantigen-binding molecule comprised of the polypeptides of SEQ ID NOs:225 and 207 is designated 10D1F.FcB (also sometimes referred to hereinas “10D1F.B”).

A modified version of 10D1 comprising the GASDALIE and LCKCsubstitutions in CH2 region was prepared and its ability to bind Fcreceptor FcγRIIIa was analysed by Bio-Layer Interferometry. The sequencefor 10D1 VH-CH1-CH2-CH3 comprising substitutions GASDALIE and LCKCcorresponding to G236A, S239D, A330L, I332E and L242C. K334C is shown inSEQ ID NO:227.

Briefly, anti-Penta-HIS (HS1K) coated biosensor tips (Pall ForteBio,USA) were used to capture His-tagged FcγRIIIa (V158) (270 nM) for 120 s.All measurements were performed at 25° C. with agitation at 1000 rpm.Association kinetic measurements for antigen binding were performed byincubating anti-HER3 antibodies at different concentrations (500 nM to15.6 nM) for 60 s, followed by a 120 s dissociation time by transferringthe biosensors into assay buffer (pH 7.2) containing wells, Sensogramswere referenced for buffer effects and then fitted using the Octet QK384user software (Pall ForteBio, USA). Kinetic responses were subjected toa global fitting using a one site binding model to obtain values forassociation (K_(on)), dissociation (K_(off)) rate constants and theequilibrium dissociation constant (K_(D)). Only curves that could bereliably fitted with the software (R²>0.90) were included in theanalysis.

The thermostability of the variant was also analysed by DifferentialScanning Fluorimetry analysis as described in Example 3.4.

FIGS. 38A and 38B show the BLI analysis and thermostability analysis,respectively, for 10D1 comprising GASDALIE and LCKC Fc substitutions.The Fc engineered 10D1 variant showed significantly improved binding toFcγRIIIa (˜9 fold increase in affinity) compared to non-Fc-engineered10D1 (see FIG. 39A) with thermal stability maintained above 60° C.

A construct for 10D1 comprising the GASD substitutions in CH2 region wasalso prepared; a sequence of 10D1 VH-CH1-CH2-CH3 comprisingsubstitutions corresponding to G236A and S239D is shown in SEQ IDNO:228.

The affinity of anti-HER3 antibody clone 10D1 ([1] of Example 2.2) andthe GASD variant thereof were analysed by Bio-Layer Interferometry foraffinity of binding to FcγRIIIa. BLI was performed as described above.

FIGS. 39A and 39B show representative sensorgrams, K_(on), K_(off) andK_(D) values. As expected, the 10D1 GASD variant (39B) displayeddramatically increased affinity for FcγRIIIa compared to 10D1 (39A).

The thermostability of the 10D1 GASD variant was also analysed byDifferential Scanning Fluorimetry analysis as described in Example 3.4.The results are shown in FIG. 40 .

Further 10D1F Fc Variant

Another antibody variant was created comprising an N297Q substitution inthe CH2 region. A representative sequence for 10D1F VH-CH1-CH2-CH3comprising the N297Q substitution is shown in SEQ ID NO:226. This‘silent form’ prevents both N-linked glycosylation of the Fc region andFc binding to Fcγ receptors and is used as a negative control.

FIGS. 41A and 41B show the binding affinity of 10D1F hIgG1 Fc variants10D1F.FcA and 10D1F.FcB to human and mouse Fc receptors, determined asdescribed above. 10D1F.FcB was found to show significantly improvedbinding to human and mouse Fcγ and FcRn receptors compared tonon-modified 10D1F.FcA or commercially available antibodies. ND=K_(D)Not Determined due to low binding affinity.

Example 8 Characterisation of Humanised and Modified Clones

8.1 Analysis of Cell Surface Antigen-Binding by Flow Cytometry

Wildtype (WT) HEK293 cells (which do not express high levels of HER3)and HEK293 cells transfected with vector encoding human HER3 (i.e.HEK293 HER O/E cells) were incubated with 10 μg/ml of humanisedanti-HER3 antibody 10D1F.FcA (10D1F), anti-HER3 antibody 10D1 (10D1P) orisotype control antibody at 4° C. for 1.5 hr. The anti-HER3 antibodyclone LJM716 (described e.g. in Garner et al., Cancer Res (2013) 73:6024-6035, and Example 3.5) was included in the analysis as a positivecontrol.

The cells were washed with buffer (PBS with 2 mM EDTA and 0.5% BSA) andresuspended in FITC-conjugated anti-FC antibody (Invitrogen, USA) at 10μg/ml for 20 min at 4° C. Cells were washed again and resuspended in 200μL of FACS flow buffer (PBS with 5 mM EDTA) for flow cytometric analysisusing MACSQuant 10 (Miltenyi Biotec, Germany). Unstained WT andtransfected HEK293 cells were included in the analysis as negativecontrols. After acquisition, all raw data were analyzed using Flowlogicsoftware. Cells were gated using forward and side scatter profilepercentage of positive cells was determined for native andoverexpressing cell populations.

The results are shown in FIGS. 42A and 42B. Anti-HER3 antibody 10D1F.FcAwas shown to bind to human HER3 with high specificity (42A). 10D1F.FcA,10D1P and LJM716 were shown to bind to human HER3-expressing cells to asimilar extent (42B).

8.2 ELISAs for Determining Antibody Specificity and Cross-Reactivity

ELISAs were used to confirm the binding specificity of the 10D1F.FcAantibody. The antibodies were analysed for their ability to bind tohuman HER3 polypeptide as well as human HER1 (EGFR) and human HER2 (SinoBiological Inc., China). Human IgG isotype and an irrelevant antigenwere included as negative controls.

ELISAs were carried out according to standard protocols. Plates werecoated with 0.1 μg/ml of target polypeptide in phosphate-buffered saline(PBS) for 16 h at 4° C. After blocking for 1 h with 1% BSA in Trisbuffer saline (TBS) at room temperature, the anti-HER3 antibody wasserially diluted with the highest concentration being 10 μg/ml, andadded to the plate. Post 1 h incubation at room temperature, plates werewashed three times with TBS containing 0.05% Tween 20 (TBS-T) and werethen incubated with a HRP-conjugated anti-His antibody (LifeTechnologies, Inc., USA) for 1 h at room temperature. After washing,plates were developed with colorimetric detection substrate3,3′,5,5′-tetramethylbenzidine (Turbo-TMB: Pierce, USA) for 10 min. Thereaction was stopped with 2M H₂SO₄, and OD was measured at 450 nM.

The results are shown in FIG. 43 . Anti-HER3 antibody 10D1F.FcA wasfound not to bind to human HER2 or human HER1 (EGFR) even at highconcentrations of the antibody.

The ability of 10D1F.FcA to bind HER4 was analysed using flow cytometry.Wildtype (WT) HEK293 cells (which do not express high levels of HER4)and HEK293 cells transfected with vector encoding human HER4 (i.e.HEK293 HER O/E cells) were incubated with 10 μg/ml of anti-HER3 antibody10D1F.FcA (10D1F) or isotype control antibody (negative control) at 4°C. for 1.5 hr. The anti-HER3 antibody clones LJM716 (described e.g. inGarner et al., Cancer Res (2013) 73: 6024-6035) and MM-121(seribantumab), as described in Example 3.5, were included in theanalysis as positive control. Also included was a commercial anti-HER4antibody (Novus, Cat: FAB11311P). Unstained HEK293 cells were includedin the analysis as negative controls.

HEK293 cells were incubated with 10 μg/ml of each antibody for 1 hour at4° C. Flow cytometry was performed as described above. Cells werecontacted with FITC-conjugated anti-FC antibody (Invitrogen, USA) at for30 min at 4° C.

The results are shown in FIG. 44 . Anti-HER3 antibody 10D1F.FcA wasfound not to bind to cell-surface expressed HER4.

In addition, antibody 10D1F.FcA was analysed for its ability to bind toHER3 polypeptide homologues from mouse, rat and monkey (Sino BiologicalInc., China), M. musculus, R. norvegicus and M. cynomolgus HER3homologues share 91.1, 91.0 and 98.9% sequence identity respectivelywith human HER3 and the HER3 signalling pathways are conserved betweenthe four species.

ELISAs were performed as above.

The results are shown in FIG. 45 . 10D1F.FcA antibody was found to bindwith high affinity to HER3 cyno, mouse, rat and human orthologs, thusdisplaying substantial cross-reactivity between species.

8.3 Global Affinity Study Using Octet QK384 System

The anti-HER3 antibody clones 10D1F.FcA and 10D1F.FcB were analysed forbinding affinity to human HER3.

Bio-Layer Interferometry (BLI) experiments were performed using theOctet QK384 system (ForteBio). Antibodies (25 nM) were coated ontoanti-Human IgG Capture (AHC) Octet sensor tips (Pall ForteBio, USA).Binding was detected using titrated HIS-tagged human HER3 in steps ofbaseline (60 s), loading (120 s), baseline2 (60 s), association (120 s),dissociation (FcA 120 s, FcB 600 s) and regeneration (15 s). Antigenconcentrations are shown in the table in FIGS. 46A and 46B. Sensorgramswere analysed as described in Example 3.3. Values were obtained forassociation (K_(on)), dissociation (K_(off)) rate constants and theequilibrium dissociation constant (K_(D)).

Representative sensorgrams for the analysis of clones 10D1F.FcA and10D1F.FcB are shown in FIGS. 46A and 46B. 10D1F.FcA binds to human HER3with a high affinity of K_(D)=72.6 pM (46A). 10D1F.FcB binds to humanHER3 with a high affinity of K_(D)=22.2 pM (46B).

8.4 Analysis of Thermostability by Differential Scanning Fluorimetry

Differential Scanning Fluorimetry was performed for antibodies 10D1F.FcAand 10D1F.FcB as described in Example 3.4.

The first derivative of the raw data obtained for Differential ScanningFluorimetry analysis of the thermostability of antibody clone 10D1F.FcAis shown in FIG. 47A. Three different samples of the antibody wereanalysed and the Tm was determined to be 70.0° C.

The first derivative of the raw data obtained for Differential ScanningFluorimetry analysis of the thermostability of antibody clone 10D1F.FcBis shown in FIG. 47B. Three different samples of the antibody wereanalysed and the Tm was determined to be 62.7° C.

8.5 Antibody Purity Analysis

The purity of antibodies 10D1F.FcA and 10D1F.FcB was analysed by sizeexclusion chromatography (SEC). 150 μg of 10D1F.FcA in 500 μl PBS pH 7.2or 150 μg of 10D1F.FcB in 500 μl PBS pH 7.45 was injected on a Superdex200 10/30 GL column in PBS running buffer at a flow rate of 0.75 min/mlor 0.5 min/ml, respectively, at room temperature and the A280 of flowthrough was recorded.

The results are shown in FIGS. 48A (10D1F.FcA) and 48B (10D1F.FcB).

8.6 Analysis of Anti-HER3 Antibody 10D1F.FcA Epitope

Anti-HER3 antibody 10D1F.FcA was analysed to determine whether itcompetes with anti-HER3 antibodies M-05-74 or M-08-11 (Roche) forbinding to HER3. Epitopes of M-05-74 and M-08-11 were both mapped to theβ-hairpin structure of the HER3 dimerisation arm located at domain II.M-08-11 does not bind to HER4 whereas M-05-74 recognises the HER4dimerisation arm. Binding of M-05-74 and M-08-11 to HER3 is ligand (NRG)independent.

BLI experiments were performed as described in Example 3.5 with onealteration: 400 nM of competing antibodies were used. The variableregions of M-05-74 and M-08-11 antibodies were coned in the PDZ vectorhaving human IgG1 and IgKappa Fc backbone.

The results of the analysis are shown in FIGS. 49A and 49B. Anti-HER310D1F.FcA antibody was found not to compete with M-05-74 or M-08-11 forbinding to HER3. 10D1F.FcA was found to bind a distinct andtopologically distant epitope of HER3 compared to M-05-74 and M-08-11.Binding of 10D1F.FcA to HER3 is ligand (NRG) independent.

Conclusions:

-   -   Binding of 10D1F.FcA to human HER3 can be achieved in a        ligand-independent manner.    -   10D1F.FcA binding epitope is distinct and topologically distant        from that of M-05-74 and M-08-11.

8.7 Inhibition of Dimerisation of HER2-HER3 and EGFR-HER3

Anti-HER3 antibody 10D1F.FcA was analysed for its ability to inhibitheterodimerisation of HER3 and HER2.

Plate-based ELISA dimerisation assays were carried out according tostandard protocols. Plates were coated with 1 μg/ml HER2-Fc protein.After blocking and washing, the plate was incubated with differentconcentrations of candidate antibodies 10D1F.FcA, MM-121, LJM716,Pertuzumab, Roche M05, Roche M08 or isotype control and constant HER3His 2 μg/ml and NRG 0.1 μg/ml for 1 hour. Plates were then washed andincubated for 1 hour with secondary anti-HIS HRP antibody. Plates werewashed, treated with TMB for 10 mins and the reaction was stopped using2M H₂SO₄ stop solution. The absorbance was read at 450 nm.

The results are shown in FIG. 52 . Anti-HER3 antibody clone 10D1F.FcAwas found to directly inhibit interaction between HER2 and HER3 in adose dependent fashion.

In another assay, inhibition of dimerization was detected usingPathHunter® Pertuzumab Bioassay Kit (DiscoverX, San Francisco, USA).HER2 and HER3 overexpressing U20S cells were thawed using 1 ml ofpre-warmed CP5 media and 5K cells were seeded per at 37° C., 5% CO₂ for4 hrs. Cells were treated with serially diluted concentrations of10D1F.FcA, Seribantumab, or Pertuzumab starting from 25 μg/ml with an8-point serial dilution. After 4 hrs incubation, 30 ng/ml ofheregulin-μ2 was added to each well and the plates were furtherincubated for 16 hrs. Following incubation, 10 μL PathHunter bioassaydetection reagent 1 was added and incubated for 15 mins at roomtemperature in the dark, followed by addition of 40 μL PathHunterbioassay detection reagent 2 which was then incubated for 60 mins atroom temperature in the dark. Plates were read using Synergy4 Biotekwith 1 second delay.

10D1F.FcA was found to have an EC₅₀ value of 3.715e-11 for inhibition ofHER2-HER3 heterodimerisation. In the same assay, the comparative EC₅₀value for Seribantumab/MM-121 was found to be 6.788e-10 and thecomparative EC₅₀ value for Pertuzumab was found to be 2.481e-10.

Anti-HER3 antibody 10D1F.FcA was analysed for its ability to inhibitheterodimerisation of EGFR and HER3.

Plate-based ELISA dimerisation assays were performed according tostandard protocols. Plate was coated with 1 μg/ml human EGFR-His. Afterblocking and washing, the plate was incubated with differentconcentrations of candidate antibodies 10D1F.FcA, MM-121, LJM716,Pertuzumab, or isotype control with constant HER3-biotin 4 μg/ml and NRG0.1 μg/ml for 1 hour. Plates were then washed and incubated for 1 hourwith secondary anti-avidin HRP antibody. Plates were washed, treatedwith TMB for 10 mins and the reaction was stopped using 2M H₂SO₄ stopsolution. The absorbance was read at 450 nm.

The results are shown in FIG. 53 . Anti-HER3 antibody clone 10D1F.FcAwas found to directly inhibit interaction between EGFR and HER3 in adose dependent fashion.

8.8 Analysis of Ability to Induce ADCC

Anti-HER3 antibody clones 10D1F.FcA and 10D1F.FcB were analysed fortheir ability to induce antibody-dependent cell-mediated cytotoxicity(ADCC).

Target cells (HEK293 overexpressing HER3) were plated in U-bottom96-well plates at a density of 20,000 cells/well. Cells were treatedwith a dilution series (50,000 ng/ml-0.18 ng/ml) of one of 10D1F.FcA,10D1F.FcB, 10D1F.FcA_N297Q (silent form), LJM-716, Seribantumab (MM-121)or left untreated, and incubated at 37° C. and 5% CO₂ for 30 min.Effector cells (Human Natural Killer Cell Line No-GFP-CD16.NK-92; 176V)were added to the plate containing target cells at a density of 60,000cells/well.

The following controls were included: Target cell maximal LDH release(target cells only), spontaneous release (target cells and effectorcells without antibody), background (culture media only). Plates werespun down and incubated at 37° C. and 5% CO₂ for 21 hrs.

LDH release assay (Pierce LDH Cytotoxicity Assay Kit); before the assay,10 μl of Lysis Buffer (10×) were added to target cell maximal LDHrelease controls and incubated at 37° C. and 5% CO₂ for 20 min. Afterincubation, plates were spun down and 50 μL of the supernatant weretransferred to clear flat-bottom 96-well plates. Reactions were startedby addition of 50 μl of LDH assay mix containing substrate to thesupernatants and incubated at 37° C. for 30 min. Reactions were stoppedby addition of 50 μl of stop solution and absorbance was recorded at 490nm and 680 nm with a BioTek Synergy HT microplate reader.

For data analysis, absorbance from test samples was corrected tobackground and spontaneous release from target cells and effector cells.Percent cytotoxicity of test samples was calculated relative to targetcell maximal LDH release controls and plot as a function of antibodyconcentration.

The results are shown in FIG. 54 . Anti-HER3 antibody 10D1F.FcB wasfound to induce potent ADCC activity against HER3 overexpressing cellsin a dose dependent fashion.

8.9 Inhibition of HER3-Mediated Signalling

Anti-HER3 antibody 10D1F.FcA was analysed for its ability to inhibitHER-3 mediated signalling in vitro in cancer cell lines.

N87, FaDu or OvCAR8 cells were seeded in wells in a 6 well plate with10% serum overnight at 37° C. with 5% CO₂. Cells were starved with 0.2%FBS culture medium for 16 hrs, and were then treated for 0.5 hours withdifferent antibodies at IC50 corresponding to the cell line. Antibodiestested were: 10D1F.FcA (10D1), Seribantumab (SBT), Elegemtumab (LJM),Pertuzumab (PTM), Cetuximab (CTX), and Trastuzumab (TZ).

Before harvesting, cells were stimulated with 100 ng/mi of NRG1. Proteinextracted from cell lines were quantified using standard Bradfordprotein assay. Protein samples (50 μg) were fractionated by SDS-PAGE andtransferred to nitrocellulose membrane. Membranes were then blocked andimmunoblotted with the indicated antibodies. The results were visualizedvia Bio-Rad Clarity Western ECL substrate. The blots were quantifiedusing densiometric analysis and data was normalised to beta actin.

The results are shown in FIGS. 55A to 55C. Anti-HER3 antibody 10D1F.FcAwas found to inhibit HER3 phosphorylation and downstream signalling inN87 (55A), FaDu (55B), OvCar8 (55C) and A549 (55D) cell lines.

For the experiments using N87 cells, A549 cells, OvCar8 cells and FaDucells total RNA was extracted at 16 hrs post antibody treatment wasanalysed to determine pathway activation based on the level ofexpression of key signal transduction pathway proteins by gene setenrichment analysis. The results of the analysis are shown in FIGS. 63Ato 63D. 10D1F.FcA was the most effective inhibitor of downstreamsignalling.

In further experiments using A549 cells, in vitro phosphorylation assayswere performed as above except that the cells were treated for 0.5 hoursor 4 hours with the different antibodies. The results are shown in FIG.64 .

Example 9 Analysis of Humanised and Modified Clones In Vitro and In Vivo

9.1 Pharmacokinetic Analysis

Mice

Female NCr nude mice approximately 6-8 weeks old were housed underspecific pathogen-free conditions and treated in compliance with theInstitutional Animal Care and Use Committee (IACUC) guidelines.

500 μg anti-HER3 antibody 10D1F.FcA or 10D1F.FcB was administered andblood was obtained from 4 mice by cardiac puncture at baseline (−2 hr),6 hr, 24 hr, 96 hr, 168 hr and 336 hr after administration. Antibody inthe serum was quantified by ELISA.

The parameters for the pharmacokinetic analysis were derived from anon-compartmental model: maximum concentration (C_(max)), AUC (0-336hr), AUC (0-infinity), Half-life (t_(1/2)), Clearance (CL), Volume ofdistribution at steady state (V_(ss)).

The results are shown in FIGS. 56A and 56B. Anti-HER3 antibody clone10D1F.FcA was found to have a half-life of 253 hours (56A) and anti-HER3antibody clone 10D1F.FcB was found to have a half-life of 273 hours(56B) in NCr nude mice.

Rats

10D1F variants were analysed to determine a single dose pharmacokineticprofile in female Sprague Dawley rats with mean weight of 320 g.

Antibody clones 10D1F.FcA and 10D1F.FcB were administered in a singledose of 4 mg (˜10 mg/kg), 10 mg (˜25 mg/kg), 40 mg (˜100 mg/kg) or 100mg (˜250 mg/kg) via tail vein slow i.v. injection. Vehicle wasadministered as a negative control. Blood from 2 rats per treatment wasobtained at baseline (−24 hr), 6 hr, 24 hr, 96 hr, 168 hr and 336 hrafter administration. Antibody in the serum was quantified by ELISA.

The parameters for the pharmacokinetic analysis were derived from anon-compartmental model: maximum concentration (C_(max)), AUC (0-336hr), AUC (0-infinity), Half-life (t_(1/2)), Clearance (CL), Volume ofdistribution at steady state (V_(d)).

The results are shown in FIGS. 57A (10 mg/kg), 57B (25 mg/kg), 57C (100mg/kg) and 57D (250 mg/kg).

9.2 Safety Immunotoxicity

The toxicological effects of 10D1F.FcA and 10D1F.FcB were analysed.

Mice

6-8 week old female BALB/c mice (20-25 g) were injectedintraperitoneally with a single dose of either 10D1F.FcA and 10D1F.FcBat one of doses: 200 ug (˜10 mg/kg), 500 ug (˜25 mg/kg), 2 mg (˜100mg/kg), or 5 mg (˜250 mg/kg), or an equal volume of PBS. 3 mice wereinjected with each treatment, 4 mice were injected with PBS control.Blood samples were obtained at 96 hours post injection and analysed forRBC indices (total RBC count, haematocrit, haemoglobin, platelet count,mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscularhaemoglobin concentration) and WBC indices (total WBC count, lymphocytecount, neutrophil count, monocyte count). Analysis was performed using aHM5 Hematology Analyser.

The results are shown in FIGS. 58A, 58B (RBC indices) and 58C (WBCindices). Anti-HER3 antibodies 10D1F.FcA and 10D1F.FcB had no effect onthe RBC indices, but were found to have an effect on the WBC indices athigher doses (10D1F.FcA 250 mg/kg, 10D1F.FcB 100 mg/kg, 10D1F.FcB 250mg/kg).

Hepatotoxicity, nephrotoxicity and pancreatic toxicity were alsoanalysed 96 hours post injection. 10D1F.FcA and 10D1F.FcB had no effecton the levels of alanine aminotransferase, alkaline phosphatase,albumin, total protein (liver indices; FIG. 58D), creatine, blood ureanitrogen, glucose or amylase (kidney and pancreatic indices; FIG. 58E).Nor did 10D1F.FcA and 10D1F.FcB have an effect on electrolyte indicessodium, potassium, calcium or phosphate (FIG. 58F).

Mice treated with 10D1F.FcA or 10D1F.FcB showed no abnormalities after96 hours in weight, behaviour, skin condition, oral examination, stooland wine examination or eye examination. Enlarged spleen (splenomegaly)approximately 1.5 times the normal size was observed in mice treatedwith higher doses: 10D1F.FcA 250 mg/kg, 10D1F.FcB 100 mg/kg, 10D1F.FcB250 mg/kg.

A further study was performed in the BALB/c mice to assess thetoxicological effects of repeat doses of 500 μg (˜25 mg/kg) 10D1F.FcA or10D1F.FcB. Antibody was administered once a week for four weeks. Bloodwas obtained 28 days after the first administration. There was no effectobserved on RBC, liver, kidney, pancreatic or electrolyte indices foreither antibody, no signs of clinical abnormalities and no differencesdetected in gross necroscopy. Total WBC count, lymphocyte count andneutrophil count was observed to be decreased in mice treated with10D1F.FcA or 10D1F.FcB but this was not considered to be toxic.

In another study, BALB/c mice were administered with a single dose of10D1F.FcA or an equal volume of PBS (vehicle control), and analysedafter 336 hours. Representative results are shown in FIGS. 69A to 69C.

Rats

6-8 week old female Sprague Dawley rats (400-450 g) were injectedintraperitoneally with a single dose of either 10D1F.FcA or 10D1F.FcBantibody at one of doses: 4 mg (˜10 mg/kg), 10 mg (˜25 mg/kg), 40 mg(˜100 mg/kg), 100 mg (˜250 mg/kg). Blood was obtained at −24 hours, 6hours, 24 hours, 96 hours, 168 hours and 336 hours. Up to 366 hours postinjection there was no effect on RBC indices, no toxic effect on WBCindices, and no effect on liver, kidney, pancreatic or electrolyteindices. There were no signs of clinical abnormalities and nodifferences detected in gross necroscopy.

Representative results obtained from rags administered with 250 mg/kg10D1F.FcA are shown in FIGS. 70A to 70C.

The absence of toxicity signals in rodent toxicology models indicatessuperior clinical safety for 10D1 and variants.

9.3 Analysis of Efficacy to Treat Cancer In Vitro

Anti-HER3 antibody 10D1F.FcA was analysed for its ability to inhibittumour growth in vitro in a number of tumour models: N87 cells (gastriccancer), HCC95 cells (lung cancer), FaDu cells (head and neck cancer),SNU-16 cells (gastric cancer). A549 cells (lung cancer), OvCar8 cells(ovarian cancer), ACHN cells (kidney cancer) and HT29 cells (colorectalcancer). 10D1F.FcA efficacy was compared to other anti HER3 antibodiesseribantumab (MM-121) and LJM-716, and other EGFR family therapiescetuximab, trastuzumab and pertuzumab.

Cells were treated with serially diluted concentrations of therapeuticantibodies, starting from 1500 ug/ml with a 9-point dilution. Cellviability was measured using CCK-8 cell proliferation assay, 3-5 dayspost treatment. The percentage of cell inhibition shown is relative tocells treated with only buffer (PBS). Data points indicates average ofthree replicates.

The results are shown in FIG. 59A to 59D. Anti-HER3 antibody 10D1F.FcAdemonstrates superior in vitro tumour inhibition in multiple tumourmodels compared to other HER3 antibodies (59A & 59B) and EGFR familytherapies (59C & 59D).

FIGS. 77A and 77B show the ability of different anti-ErbB antibodies toinhibit proliferation of different cancer cell lines in vitro, at theC_(max) concentration they achieve in mice administered IP with 25 mg/kgof the relevant antibody. 10D1F.FcA displays outstanding ability toinhibit growth of a wide variety of different cancer cell types.

9.4 Analysis of Efficacy to Treat Cancer In Vivo

Anti-HER3 antibody clones 10D1F.FcA and 10D1F.FcB were assessed fortheir effect on tumour growth in in vivo cancer models.

9.4.1 A549 Model

Tumour cells were inserted subcutaneously into the right flanks offemale NCr nude mice. Antibodies (25 mg/kg 10D1F.FcA, 10D1F.FcB,Cetuximab, LJM-716 or MM-121; n=6 for each treatment) or vehicle (n=8)were administered biweekly for six weeks.

The results are shown in FIG. 60 . Anti-HER3 antibody clones 10D1F.FcAand 10D1F.FcB both displayed potent efficacy in the A549 model of lungcarcinoma. 10D1F.FcB was found to be particularly potent and madetumours regress.

9.4.2 FaDu Model

Tumour cells were inserted with matrigel subcutaneously into the rightflanks of female NCr nude mice. Antibodies (10 and 25 mg/kg 10D1F.FcAand 10D1F.FcB, or 25 mg/kg of Cetuximab, Trastuzumab, Pertuzumab,LJM-716 or MM-121; n=6 for each treatment) or vehicle (n=6) wereadministered once a week for six weeks.

The results are shown in FIG. 61 . Anti-HER3 antibody clones 10D1F.FcAand 10D1F.FcB were both found to be effective to prevent tumour growthin the FaDu model of head and neck cancer.

9.4.3 OvCar8 Model

Tumour cells were inserted with matrigel subcutaneously into the rightflanks of female NCr nude mice. Antibodies (10 and 25 mg/kg 10D1F.FcA,or 25 mg/kg Cetuximab, LJM-716 or MM-121; n=6) or vehicle (n=6) wereadministered once a week for six weeks.

The results are shown in FIG. 62 . Anti-HER3 antibody clone 10D1F.FcAwas found to be effective at reducing tumour volume at higher dose.

9.4.4 N87 Model

Tumour cells are inserted with matrigel subcutaneously into the rightflanks of female NCr nude mice. Antibodies (25 mg/kg 10D1F.FcA, or 50mg/kg of Trastuzumab, LJM-716 or MM-121; n=6 for each treatment) orvehicle (n=6) were administered biweekly for six weeks.

The results are shown in FIG. 74 . Anti-HER3 antibody 10D1F.FcA and wasfound to be effective to prevent tumour growth in the N87 model ofgastric cancer.

Example 10 Analysis of Inhibition of Proliferation of BRAFV600E MutantThyroid Cancer Cell Lines

The following cell fines were investigated:

Cell Line Type of Cancer Mutation SW1736 Anaplastic thyroid cancer BRAFV600E BHT101 Anaplastic thyroid cancer BRAF V600E BCPAP Papillarythyroid cancer BRAF V600E and p53 mutation

The cells were investigated for surface expression of EGFR familymembers by flow cytometry. Briefly, 300,000 cells were incubated with 20μg/ml of 10D1F.FcA, cetuximab or trastuzumab for 1 hr at 4° C.Alexafluor 488-conjugated anti-human antibody was used at 10 μg/ml as asecondary antibody (40 min at 4° C.).

The results are shown in FIG. 66A to 66C. SW1736, BHT101 and BCPAP cellswere shown to express EGFR, HER2 and HER3.

The inventors investigated the ability of different HER3-bindingantibodies to inhibit in vitro proliferation of different thyroid cancercell lines harbouring the V600E BRAF mutation.

Briefly, cells of the different cell lines were seeded at a density of1.5×10⁵ cells/well, and treated the next day with a 10 point serialdilution starting from 1000 μg/ml of 10D1F.FcA, seribantumab, LJM-716,pertuzumab or isotype control antibody. After 3 days, proliferation wasmeasured using a CCK-8 cell proliferation assay. Percent inhibition ofproliferation was calculated relative to cells treated with an equalvolume of PBS instead of antibodies.

The results are shown in FIGS. 67A to 67C. 10D1F.FcA was found to bemore effective at inhibiting proliferation of cell lines harbouring BRAFV600E mutation than any other of the anti-HER3 antibodies analysed.

In further experiments, the ability of a combination of 10D1F.FcA andvemurafenib to inhibit in vitro proliferation of different thyroidcancer cell lines harbouring the V600E BRAF mutation was investigated.

Cells were seeded at a density of 1.5×10⁵ cells/well, and treated thenext day with a 10 point serial dilution starting from 1000 μg/ml of10D1F.FcA or isotype control antibody, in the presence or absence of 200nM vemurafenib. After 3 days, proliferation was measured using a CCK-8cell proliferation assay. Percent inhibition of proliferation wascalculated relative to cells treated with an equal volume of PBS insteadof antibodies.

The results are shown in FIGS. 68A to 68C. 10D1F.FcA was found toenhance the ability of vemurafenib to inhibit proliferation of SW1736and BHT101 cells, which are susceptible to vemurafenib. 10D1F.FcA wasalso found to be a potent inhibitor of proliferation ofvemurafenib-resistant BCPAP cells.

Example 11 Analysis of Inhibition of HER3-Mediated Signalling In Vivo

The inventors investigated the ability of 10D1F.FcA to inhibitHER3-mediated signalling in vivo.

1×10⁶ FaDu or OvCar8 cells were introduced subcutaneously into NCr nudemice, to establish ectopic xenograft tumors.

Once tumors had reached a volume of greater than 100 mm³, mice were withtreated by biweekly intraperitoneal injection of 10D1F.FcA at a dose of25 mg/kg, or an equal volume of vehicle (control). After 4 weeks, tumorswere harvested. Protein extracts were prepared from the tumors andquantified via Bradford assay, 50 μg samples were fractionated bySDS-PAGE, and analysed by western blot using antibodies in order todetermine in vivo phosphorylation of HER3 and AKT, as described inExample 4.3.

The results are shown in FIG. 71 . 10D1F.FcA was found to inhibitphosphorylation of HER3 and AKT in tumor cells in vivo.

Example 12 Analysis of Internalisation of Anti-HER3 Antibodies

The inventors investigated internalisation of anti-HER3 antibodies byHER3-expressing cells.

Briefly, 100,000 HEK293 cells engineered to express HER3, HCC95, N87 orOVCAR8 cells were seeded in wells of 96-well tissue culture plates andcultured overnight at 37° C. in 5% CO₂. Cells were then treated with 120nM of 10D1F.FcA, LJM-716, seribantumab or trastuzumab, and 360 nM ofpHrodo iFL Green reagent, and incubated at 37° C. in 5% CO₂. The cellsin culture were imaged every 30 min for 24 hours, in 4 different fieldsof each well. The maximum signal intensity in the FITC channel of eachfield was quantified at 24 hours.

The results are shown in FIG. 72 . Modest to moderate internalization ofLJM-716 and seribantumab was observed in OvCar8 cells, whereas modestinternalization of trastuzumab was observed in N87 cells.

No significant internalization of 10D1F.FcA was observed in HCC95, N87,or OvCar8 cells.

As expected, significant internalization of 10D1F.FcA, LJM-716, andseribantumab was observed in HEK293 cells overexpressing HER3.

In further experiments, antibody internalisation was investigated byflow cytometry.

N37 cells were seeded in wells of 96-well tissue culture plates at adensity of 50,000 cells/well, and allowed to adhere overnight (37° C.,5% CO₂). 10D1F.FcA or trastuzumab were mixed with labelling reagent, andthe labelled complexes were added to cells. Samples were harvested at 0min, 10 min, 30 min, 1 hour, 2 hour and 4.5 hour time points, byaspiration of cell culture medium, washing with PBS and treatment withaccutase. Accutase activity was neutralised, and cells were resuspendedin FACs buffer and analysed by flow cytometry.

The results are shown in FIGS. 73A and 73B. The cells displayed minimalinternalisation of 10D1F.FcA. By contrast, substantial internalisationof anti-HER2 antibody trastuzumab was observed.

Example 13 Use of HER3-Binding Antibodies in Immunohistochemistry

Anti-HER3 antibody 10D1F in mIgG2a format was evaluated for its abilityto be used in immunohistochemistry for the detection of human HER3protein.

Processing of sections was performed using Bond reagents (LeicaBiosystems). Arrays of commercially-available frozen tissue sectionswere obtained. Slides were dried in a desiccator for 10 min and thensubjected to the following treatments, with water washes and/or TBS-Trinses between steps: (i) fixation by treatment with 100% acetone for 10min at room temperature; (ii) endogenous peroxidase blocking bytreatment with 3% (v/v) H₂O₂ for 15 min at room temperature; (iii)blocking by treatment with 10% goat serum for 30 min at roomtemperature, (iv) incubation with 10D1F-mIgG2a at 1:250 dilution of a6.2 mg/ml solution overnight at 4° C., (v) incubation with HRP-polymerconjugated goat anti-mouse antibody for 30 min at room temperature, and(vi) development with Bond Mixed DAB Refine for 5 min at roomtemperature, followed by rinsing with deionised water and 1× Bond Washto stop the reaction.

Slides were then dehydrated, mounted in synthetic mounting media andscanned with high resolution.

The results are shown in FIGS. 75A and 75B. 10D1F preferentially stainedmalignant human tissue sections, with low cross-reactivity to normaltissue.

In further experiments, an A549 xenograft tumor was harvested in coldPBS, embedded in OCT cryoembedding medium, frozen in dry-ice and storedat −80° C. 10 μm sections were obtained using a cryostat.

Slides were dried in a desiccator for 10 min and then subjected to thefollowing treatments, with water washes and/or TBS-T rinses betweensteps: (i) fixation by treatment with 100% acetone for 10 min at roomtemperature; (ii) endogenous peroxidase blocking by treatment with 3%(v/v) H₂O₂ for 15 min at room temperature; (iii) blocking by treatmentwith 10% goat serum for 30 min at room temperature, (iv) incubation with10D1F.FcA at 1:50 dilution of 8.8 mg/ml solution, or with 1:200 dilutionof Sino Biological rabbit anti-HER3 (Cat. No. 10201-T24) overnight at 4°C., (v) incubation with Invitrogen F(ab′)2-Goat anti-Human IgG (H+L) HRP(A24470) (1:500), or HRP-polymer conjugated goat anti-rabbit antibodyfor 30 min at room temperature, and (vi) development with Bond Mixed DABRefine for 5 min at room temperature, followed by rinsing with deionisedwater and 1× Bond Wash to stop the reaction.

Slides were then counterstained with haematoxylin, dehydrated, mountedin synthetic mounting media and scanned with high resolution.

The results are shown in FIG. 76 . 10D1F.FcA displayed specific membraneand cytoplasmic staining of A549 tumor xenograft cryosections.

The invention claimed is:
 1. An antigen-binding molecule comprising aheavy chain variable (VH) region and a light chain variable (VL) region,wherein the antigen-binding molecule specifically binds to HER3, whereinbinding to HER3 comprises contact with one or more amino acid residuesof the region of HER3 shown in SEQ ID NO:229, and wherein theantigen-binding molecule comprises: a VH region incorporating thefollowing CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO:43HC-CDR2 having the amino acid sequence of SEQ ID NO:46 HC-CDR3 havingthe amino acid sequence of SEQ ID NO:51; and a VL region incorporatingthe following CDRs: LC-CDR1 having the amino acid sequence of SEQ IDNO:91 LC-CDR2 having the amino acid sequence of SEQ ID NO:94 LC-CDR3having the amino acid sequence of SEQ ID NO:99.
 2. The antigen-bindingmolecule according to claim 1, wherein the antigen-binding moleculecomprises: (a) a VH region having the following CDRs: HC-CDR1 having theamino acid sequence of SEQ ID NO:41 HC-CDR2 having the amino acidsequence of SEQ ID NO:44 HC-CDR3 having the amino acid sequence of SEQID NO:47; and a VL region having the following CDRs: LC-CDR1 having theamino acid sequence of SEQ ID NO:88 LC-CDR2 having the amino acidsequence of SEQ ID NO:92 LC-CDR3 having the amino acid sequence of SEQID NO:95; or (b) a VH region having the following CDRs: HC-CDR1 havingthe amino acid sequence of SEQ ID NO:41 HC-CDR2 having the amino acidsequence of SEQ ID NO:44 HC-CDR3 having the amino acid sequence of SEQID NO:47; and a VL region having the following CDRs: LC-CDR1 having theamino acid sequence of SEQ ID NO:89 LC-CDR2 having the amino acidsequence of SEQ ID NO:92 LC-CDR3 having the amino acid sequence of SEQID NO:95; or (c) a VH region having the following CDRs: HC-CDR1 havingthe amino acid sequence of SEQ ID NO:41 HC-CDR2 having the amino acidsequence of SEQ ID NO:44 HC-CDR3 having the amino acid sequence of SEQID NO:47; and a VL region having the following CDRs: LC-CDR1 having theamino acid sequence of SEQ ID NO:90 LC-CDR2 having the amino acidsequence of SEQ ID NO:92 LC-CDR3 having the amino acid sequence of SEQID NO:96; or (d) a VH region having the following CDRs: HC-CDR1 havingthe amino acid sequence of SEQ ID NO:41 HC-CDR2 having the amino acidsequence of SEQ ID NO:45 HC-CDR3 having the amino acid sequence of SEQID NO:47; and a VL region having the following CDRs: LC-CDR1 having theamino acid sequence of SEQ ID NO:88 LC-CDR2 having the amino acidsequence of SEQ ID NO:93 LC-CDR3 having the amino acid sequence of SEQID NO:95; or (e) a VH region having the following CDRs: HC-CDR1 havingthe amino acid sequence of SEQ ID NO:41 HC-CDR2 having the amino acidsequence of SEQ ID NO:45 HC-CDR3 having the amino acid sequence of SEQID NO:49; and a VL region having the following CDRs: LC-CDR1 having theamino acid sequence of SEQ ID NO:88 LC-CDR2 having the amino acidsequence of SEQ ID NO:93 LC-CDR3 having the amino acid sequence of SEQID NO:95; or (f) a VH region having the following CDRs: HC-CDR1 havingthe amino acid sequence of SEQ ID NO:41 HC-CDR2 having the amino acidsequence of SEQ ID NO:45 HC-CDR3 having the amino acid sequence of SEQID NO:50; and a VL region having the following CDRs: LC-CDR1 having theamino acid sequence of SEQ ID NO:88 LC-CDR2 having the amino acidsequence of SEQ ID NO:93 LC-CDR3 having the amino acid sequence of SEQID NO:95; or (g) a VH region having the following CDRs: HC-CDR1 havingthe amino acid sequence of SEQ ID NO:41 HC-CDR2 having the amino acidsequence of SEQ ID NO:45 HC-CDR3 having the amino acid sequence of SEQID NO:48; and a VL region having the following CDRs: LC-CDR1 having theamino acid sequence of SEQ ID NO:88 LC-CDR2 having the amino acidsequence of SEQ ID NO:92 LC-CDR3 having the amino acid sequence of SEQID NO:95; or (h) a VH region having the following CDRs: HC-CDR1 havingthe amino acid sequence of SEQ ID NO:41 HC-CDR2 having the amino acidsequence of SEQ ID NO:45 HC-CDR3 having the amino acid sequence of SEQID NO:48; and a VL region having the following CDRs: LC-CDR1 having theamino acid sequence of SEQ ID NO:88 LC-CDR2 having the amino acidsequence of SEQ ID NO:92 LC-CDR3 having the amino acid sequence of SEQID NO:97; or (i) a VH region having the following CDRs: HC-CDR1 havingthe amino acid sequence of SEQ ID NO:42 HC-CDR2 having the amino acidsequence of SEQ ID NO:45 HC-CDR3 having the amino acid sequence of SEQID NO:48; and a VL region having the following CDRs: LC-CDR1 having theamino acid sequence of SEQ ID NO:88 LC-CDR2 having the amino acidsequence of SEQ ID NO:92 LC-CDR3 having the amino acid sequence of SEQID NO:95; or (j) a VH region having the following CDRs: HC-CDR1 havingthe amino acid sequence of SEQ ID NO:41 HC-CDR2 having the amino acidsequence of SEQ ID NO:44 HC-CDR3 having the amino acid sequence of SEQID NO:47; and a VL region having the following CDRs: LC-CDR1 having theamino acid sequence of SEQ ID NO:88 LC-CDR2 having the amino acidsequence of SEQ ID NO:92 LC-CDR3 having the amino acid sequence of SEQID NO:98.
 3. The antigen-binding molecule according to claim 1, whereinthe antigen-binding molecule comprises: (a) a VH region comprising anamino acid sequence having at least 70% sequence identity to the aminoacid sequence of SEQ ID NO:24; and a VL region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:74; or (b) a VH region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:25; and a VL region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:75; or (c) a VH region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:26; and a VL region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:76; or (d) a VH region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:27; and a VL region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:77; or (e) a VH region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:28; and a VL region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:78; or (f) a VH region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:29; and a VL region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:78; or (g) a VH region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:30; and a VL region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:78; or (h) a VH region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:31; and a VL region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:79; or (i) a VH region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:32; and a VL region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:79; or (j) a VH region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:33; and a VL region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:80; or (k) a VH region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:34; and a VL region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:81; or (l) a VH region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:35; and a VL region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:82; or (m) a VH region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:36; and a VL region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:83; or (n) a VH region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:37; and a VL region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:84; or (o) a VH region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:38; and a VL region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:85; or (p) a VH region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:39; and a VL region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:86; or (q) a VH region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:40; and a VL region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:87.
 4. A nucleic acid, or a plurality of nucleicacids, encoding an antigen-binding molecule comprising a heavy chainvariable (VH) region and a light chain variable (VL) region, wherein theantigen-binding molecule specifically binds to HER3, wherein binding toHER3 comprises contact with one or more amino acid residues of theregion of HER3 shown in SEQ ID NO:229, and wherein the antigen-bindingmolecule comprises: a VH region incorporating the following CDRs:HC-CDR1 having the amino acid sequence of SEQ ID NO:43 HC-CDR2 havingthe amino acid sequence of SEQ ID NO:46 HC-CDR3 having the amino acidsequence of SEQ ID NO:51; and a VL region incorporating the followingCDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO:91 LC-CDR2having the amino acid sequence of SEQ ID NO:94 LC-CDR3 having the aminoacid sequence of SEQ ID NO:99.
 5. The nucleic acid or plurality ofnucleic acids according to claim 4, wherein the antigen-binding moleculecomprises: (a) a VH region having the following CDRs: HC-CDR1 having theamino acid sequence of SEQ ID NO:41 HC-CDR2 having the amino acidsequence of SEQ ID NO:44 HC-CDR3 having the amino acid sequence of SEQID NO:47; and a VL region having the following CDRs: LC-CDR1 having theamino acid sequence of SEQ ID NO:88 LC-CDR2 having the amino acidsequence of SEQ ID NO:92 LC-CDR3 having the amino acid sequence of SEQID NO:95; or (b) a VH region having the following CDRs: HC-CDR1 havingthe amino acid sequence of SEQ ID NO:41 HC-CDR2 having the amino acidsequence of SEQ ID NO:44 HC-CDR3 having the amino acid sequence of SEQID NO:47; and a VL region having the following CDRs: LC-CDR1 having theamino acid sequence of SEQ ID NO:89 LC-CDR2 having the amino acidsequence of SEQ ID NO:92 LC-CDR3 having the amino acid sequence of SEQID NO:95; or (c) a VH region having the following CDRs: HC-CDR1 havingthe amino acid sequence of SEQ ID NO:41 HC-CDR2 having the amino acidsequence of SEQ ID NO:44 HC-CDR3 having the amino acid sequence of SEQID NO:47; and a VL region having the following CDRs: LC-CDR1 having theamino acid sequence of SEQ ID NO:90 LC-CDR2 having the amino acidsequence of SEQ ID NO:92 LC-CDR3 having the amino acid sequence of SEQID NO:96; or (d) a VH region having the following CDRs: HC-CDR1 havingthe amino acid sequence of SEQ ID NO:41 HC-CDR2 having the amino acidsequence of SEQ ID NO:45 HC-CDR3 having the amino acid sequence of SEQID NO:47; and a VL region having the following CDRs: LC-CDR1 having theamino acid sequence of SEQ ID NO:88 LC-CDR2 having the amino acidsequence of SEQ ID NO:93 LC-CDR3 having the amino acid sequence of SEQID NO:95; or (e) a VH region having the following CDRs: HC-CDR1 havingthe amino acid sequence of SEQ ID NO:41 HC-CDR2 having the amino acidsequence of SEQ ID NO:45 HC-CDR3 having the amino acid sequence of SEQID NO:49; and a VL region having the following CDRs: LC-CDR1 having theamino acid sequence of SEQ ID NO:88 LC-CDR2 having the amino acidsequence of SEQ ID NO:93 LC-CDR3 having the amino acid sequence of SEQID NO:95; or (f) a VH region having the following CDRs: HC-CDR1 havingthe amino acid sequence of SEQ ID NO:41 HC-CDR2 having the amino acidsequence of SEQ ID NO:45 HC-CDR3 having the amino acid sequence of SEQID NO:50; and a VL region having the following CDRs: LC-CDR1 having theamino acid sequence of SEQ ID NO:88 LC-CDR2 having the amino acidsequence of SEQ ID NO:93 LC-CDR3 having the amino acid sequence of SEQID NO:95; or (g) a VH region having the following CDRs: HC-CDR1 havingthe amino acid sequence of SEQ ID NO:41 HC-CDR2 having the amino acidsequence of SEQ ID NO:45 HC-CDR3 having the amino acid sequence of SEQID NO:48; and a VL region having the following CDRs: LC-CDR1 having theamino acid sequence of SEQ ID NO:88 LC-CDR2 having the amino acidsequence of SEQ ID NO:92 LC-CDR3 having the amino acid sequence of SEQID NO:95; or (h) a VH region having the following CDRs: HC-CDR1 havingthe amino acid sequence of SEQ ID NO:41 HC-CDR2 having the amino acidsequence of SEQ ID NO:45 HC-CDR3 having the amino acid sequence of SEQID NO:48; and a VL region having the following CDRs: LC-CDR1 having theamino acid sequence of SEQ ID NO:88 LC-CDR2 having the amino acidsequence of SEQ ID NO:92 LC-CDR3 having the amino acid sequence of SEQID NO:97; or (i) a VH region having the following CDRs: HC-CDR1 havingthe amino acid sequence of SEQ ID NO:42 HC-CDR2 having the amino acidsequence of SEQ ID NO:45 HC-CDR3 having the amino acid sequence of SEQID NO:48; and a VL region having the following CDRs: LC-CDR1 having theamino acid sequence of SEQ ID NO:88 LC-CDR2 having the amino acidsequence of SEQ ID NO:92 LC-CDR3 having the amino acid sequence of SEQID NO:95; or (j) a VH region having the following CDRs: HC-CDR1 havingthe amino acid sequence of SEQ ID NO:41 HC-CDR2 having the amino acidsequence of SEQ ID NO:44 HC-CDR3 having the amino acid sequence of SEQID NO:47; and a VL region having the following CDRs: LC-CDR1 having theamino acid sequence of SEQ ID NO:88 LC-CDR2 having the amino acidsequence of SEQ ID NO:92 LC-CDR3 having the amino acid sequence of SEQID NO:98.
 6. The nucleic acid or plurality of nucleic acids according toclaim 4, wherein the antigen-binding molecule comprises: (a) a VH regioncomprising an amino acid sequence having at least 70% sequence identityto the amino acid sequence of SEQ ID NO:24; and a VL region comprisingan amino acid sequence having at least 70% sequence identity to theamino acid sequence of SEQ ID NO:74; or (b) a VH region comprising anamino acid sequence having at least 70% sequence identity to the aminoacid sequence of SEQ ID NO:25; and a VL region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:75; or (c) a VH region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:26; and a VL region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:76; or (d) a VH region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:27; and a VL region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:77; or (e) a VH region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:28; and a VL region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:78; or (f) a VH region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:29; and a VL region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:78; or (g) a VH region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:30; and a VL region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:78; or (h) a VH region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:31; and a VL region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:79; or (i) a VH region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:32; and a VL region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:79; or (j) a VH region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:33; and a VL region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:80; or (k) a VH region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:34; and a VL region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:81; or (l) a VH region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:35; and a VL region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:82; or (m) a VH region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:36; and a VL region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:83; or (n) a VH region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:37; and a VL region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:84; or (o) a VH region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:38; and a VL region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:85; or (p) a VH region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:39; and a VL region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:86; or (q) a VH region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:40; and a VL region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:87.
 7. A cell comprising a nucleic acid, or aplurality of nucleic acids, encoding an antigen-binding moleculecomprising a heavy chain variable (VH) region and a light chain variable(VL) region, wherein the antigen-binding molecule specifically binds toHER3, wherein binding to HER3 comprises contact with one or more aminoacid residues of the region of HER3 shown in SEQ ID NO:229, and whereinthe antigen-binding molecule comprises: a VH region incorporating thefollowing CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO:43HC-CDR2 having the amino acid sequence of SEQ ID NO:46 HC-CDR3 havingthe amino acid sequence of SEQ ID NO:51; and a VL region incorporatingthe following CDRs: LC-CDR1 having the amino acid sequence of SEQ IDNO:91 LC-CDR2 having the amino acid sequence of SEQ ID NO:94 LC-CDR3having the amino acid sequence of SEQ ID NO:99.
 8. The cell according toclaim 7, wherein the antigen-binding molecule comprises: (a) a VH regionhaving the following CDRs: HC-CDR1 having the amino acid sequence of SEQID NO:41 HC-CDR2 having the amino acid sequence of SEQ ID NO:44 HC-CDR3having the amino acid sequence of SEQ ID NO:47; and a VL region havingthe following CDRs: LC-CDR1 having the amino acid sequence of SEQ IDNO:88 LC-CDR2 having the amino acid sequence of SEQ ID NO:92 LC-CDR3having the amino acid sequence of SEQ ID NO:95; or (b) a VH regionhaving the following CDRs: HC-CDR1 having the amino acid sequence of SEQID NO:41 HC-CDR2 having the amino acid sequence of SEQ ID NO:44 HC-CDR3having the amino acid sequence of SEQ ID NO:47; and a VL region havingthe following CDRs: LC-CDR1 having the amino acid sequence of SEQ IDNO:89 LC-CDR2 having the amino acid sequence of SEQ ID NO:92 LC-CDR3having the amino acid sequence of SEQ ID NO:95; or (c) a VH regionhaving the following CDRs: HC-CDR1 having the amino acid sequence of SEQID NO:41 HC-CDR2 having the amino acid sequence of SEQ ID NO:44 HC-CDR3having the amino acid sequence of SEQ ID NO:47; and a VL region havingthe following CDRs: LC-CDR1 having the amino acid sequence of SEQ IDNO:90 LC-CDR2 having the amino acid sequence of SEQ ID NO:92 LC-CDR3having the amino acid sequence of SEQ ID NO:96; or (d) a VH regionhaving the following CDRs: HC-CDR1 having the amino acid sequence of SEQID NO:41 HC-CDR2 having the amino acid sequence of SEQ ID NO:45 HC-CDR3having the amino acid sequence of SEQ ID NO:47; and a VL region havingthe following CDRs: LC-CDR1 having the amino acid sequence of SEQ IDNO:88 LC-CDR2 having the amino acid sequence of SEQ ID NO:93 LC-CDR3having the amino acid sequence of SEQ ID NO:95; or (e) a VH regionhaving the following CDRs: HC-CDR1 having the amino acid sequence of SEQID NO:41 HC-CDR2 having the amino acid sequence of SEQ ID NO:45 HC-CDR3having the amino acid sequence of SEQ ID NO:49; and (f) a VH regionhaving the following CDRs: HC-CDR1 having the amino acid sequence of SEQID NO:41 HC-CDR2 having the amino acid sequence of SEQ ID NO:45 HC-CDR3having the amino acid sequence of SEQ ID NO:50; and a VL region havingthe following CDRs: LC-CDR1 having the amino acid sequence of SEQ IDNO:88 LC-CDR2 having the amino acid sequence of SEQ ID NO:93 LC-CDR3having the amino acid sequence of SEQ ID NO:95; or (g) a VH regionhaving the following CDRs: HC-CDR1 having the amino acid sequence of SEQID NO:41 HC-CDR2 having the amino acid sequence of SEQ ID NO:45 HC-CDR3having the amino acid sequence of SEQ ID NO:48; and a VL region havingthe following CDRs: LC-CDR1 having the amino acid sequence of SEQ IDNO:88 LC-CDR2 having the amino acid sequence of SEQ ID NO:92 LC-CDR3having the amino acid sequence of SEQ ID NO:95; or (h) a VH regionhaving the following CDRs: HC-CDR1 having the amino acid sequence of SEQID NO:41 HC-CDR2 having the amino acid sequence of SEQ ID NO:45 HC-CDR3having the amino acid sequence of SEQ ID NO:48; and a VL region havingthe following CDRs: LC-CDR1 having the amino acid sequence of SEQ IDNO:88 LC-CDR2 having the amino acid sequence of SEQ ID NO:92 LC-CDR3having the amino acid sequence of SEQ ID NO:97; or (i) a VH regionhaving the following CDRs: HC-CDR1 having the amino acid sequence of SEQID NO:42 HC-CDR2 having the amino acid sequence of SEQ ID NO:45 HC-CDR3having the amino acid sequence of SEQ ID NO:48; and a VL region havingthe following CDRs: LC-CDR1 having the amino acid sequence of SEQ IDNO:88 LC-CDR2 having the amino acid sequence of SEQ ID NO:92 LC-CDR3having the amino acid sequence of SEQ ID NO:95; or (j) a VH regionhaving the following CDRs: HC-CDR1 having the amino acid sequence of SEQID NO:41 HC-CDR2 having the amino acid sequence of SEQ ID NO:44 HC-CDR3having the amino acid sequence of SEQ ID NO:47; and a VL region havingthe following CDRs: LC-CDR1 having the amino acid sequence of SEQ IDNO:88 LC-CDR2 having the amino acid sequence of SEQ ID NO:92 LC-CDR3having the amino acid sequence of SEQ ID NO:98.
 9. The cell according toclaim 7, wherein the antigen-binding molecule comprises: (a) a VH regioncomprising an amino acid sequence having at least 70% sequence identityto the amino acid sequence of SEQ ID NO:24; and a VL region comprisingan amino acid sequence having at least 70% sequence identity to theamino acid sequence of SEQ ID NO:74; or (b) a VH region comprising anamino acid sequence having at least 70% sequence identity to the aminoacid sequence of SEQ ID NO:25; and a VL region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:75; or (c) a VH region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:26; and a VL region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:76; or (d) a VH region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:27; and a VL region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:77; or (e) a VH region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:28; and a VL region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:78; or (f) a VH region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:29; and a VL region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:78; or (g) a VH region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:30; and a VL region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:78; or (h) a VH region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:31; and a VL region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:79; or (i) a VH region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:32; and a VL region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:79; or (j) a VH region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:33; and a VL region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:80; or (k) a VH region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:34; and a VL region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:81; or (l) a VH region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:35; and a VL region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:82; or (m) a VH region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:36; and a VL region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:83; or (n) a VH region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:37; and a VL region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:84; or (o) a VH region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:38; and a VL region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:85; or (p) a VH region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:39; and a VL region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:86; or (q) a VH region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:40; and a VL region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:87.
 10. A method of treating a cancer in asubject, comprising administering to a subject a therapeuticallyeffective amount of an antigen-binding molecule comprising a heavy chainvariable (VH) region and a light chain variable (VL) region, wherein theantigen-binding molecule specifically binds to HER3, wherein binding toHER3 comprises contact with one or more amino acid residues of theregion of HER3 shown in SEQ ID NO:229, and wherein the antigen-bindingmolecule comprises: a VH region incorporating the following CDRs:HC-CDR1 having the amino acid sequence of SEQ ID NO:43 HC-CDR2 havingthe amino acid sequence of SEQ ID NO:46 HC-CDR3 having the amino acidsequence of SEQ ID NO:51; and a VL region incorporating the followingCDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO:91 LC-CDR2having the amino acid sequence of SEQ ID NO:94 LC-CDR3 having the aminoacid sequence of SEQ ID NO:99.
 11. The method according to claim 10,wherein the antigen-binding molecule comprises: (a) a VH region havingthe following CDRs: HC-CDR1 having the amino acid sequence of SEQ IDNO:41 HC-CDR2 having the amino acid sequence of SEQ ID NO:44 HC-CDR3having the amino acid sequence of SEQ ID NO:47; and a VL region havingthe following CDRs: LC-CDR1 having the amino acid sequence of SEQ IDNO:88 LC-CDR2 having the amino acid sequence of SEQ ID NO:92 LC-CDR3having the amino acid sequence of SEQ ID NO:95; or (b) a VH regionhaving the following CDRs: HC-CDR1 having the amino acid sequence of SEQID NO:41 HC-CDR2 having the amino acid sequence of SEQ ID NO:44 HC-CDR3having the amino acid sequence of SEQ ID NO:47; and a VL region havingthe following CDRs: LC-CDR1 having the amino acid sequence of SEQ IDNO:89 LC-CDR2 having the amino acid sequence of SEQ ID NO:92 LC-CDR3having the amino acid sequence of SEQ ID NO:95; or (c) a VH regionhaving the following CDRs: HC-CDR1 having the amino acid sequence of SEQID NO:41 HC-CDR2 having the amino acid sequence of SEQ ID NO:44 HC-CDR3having the amino acid sequence of SEQ ID NO:47; and a VL region havingthe following CDRs: LC-CDR1 having the amino acid sequence of SEQ IDNO:90 LC-CDR2 having the amino acid sequence of SEQ ID NO:92 LC-CDR3having the amino acid sequence of SEQ ID NO:96; or (d) a VH regionhaving the following CDRs: HC-CDR1 having the amino acid sequence of SEQID NO:41 HC-CDR2 having the amino acid sequence of SEQ ID NO:45 HC-CDR3having the amino acid sequence of SEQ ID NO:47; and a VL region havingthe following CDRs: LC-CDR1 having the amino acid sequence of SEQ IDNO:88 LC-CDR2 having the amino acid sequence of SEQ ID NO:93 LC-CDR3having the amino acid sequence of SEQ ID NO:95; or (e) a VH regionhaving the following CDRs: HC-CDR1 having the amino acid sequence of SEQID NO:41 HC-CDR2 having the amino acid sequence of SEQ ID NO:45 HC-CDR3having the amino acid sequence of SEQ ID NO:49; and a VL region havingthe following CDRs: LC-CDR1 having the amino acid sequence of SEQ IDNO:88 LC-CDR2 having the amino acid sequence of SEQ ID NO:93 LC-CDR3having the amino acid sequence of SEQ ID NO:95; or (f) a VH regionhaving the following CDRs: HC-CDR1 having the amino acid sequence of SEQID NO:41 HC-CDR2 having the amino acid sequence of SEQ ID NO:45 HC-CDR3having the amino acid sequence of SEQ ID NO:50; and a VL region havingthe following CDRs: LC-CDR1 having the amino acid sequence of SEQ IDNO:88 LC-CDR2 having the amino acid sequence of SEQ ID NO:93 LC-CDR3having the amino acid sequence of SEQ ID NO:95; or (g) a VH regionhaving the following CDRs: HC-CDR1 having the amino acid sequence of SEQID NO:41 HC-CDR2 having the amino acid sequence of SEQ ID NO:45 HC-CDR3having the amino acid sequence of SEQ ID NO:48; and a VL region havingthe following CDRs: LC-CDR1 having the amino acid sequence of SEQ IDNO:88 LC-CDR2 having the amino acid sequence of SEQ ID NO:92 LC-CDR3having the amino acid sequence of SEQ ID NO:95; or (h) a VH regionhaving the following CDRs: HC-CDR1 having the amino acid sequence of SEQID NO:41 HC-CDR2 having the amino acid sequence of SEQ ID NO:45 HC-CDR3having the amino acid sequence of SEQ ID NO:48; and a VL region havingthe following CDRs: LC-CDR1 having the amino acid sequence of SEQ IDNO:88 LC-CDR2 having the amino acid sequence of SEQ ID NO:92 LC-CDR3having the amino acid sequence of SEQ ID NO:97; or (i) a VH regionhaving the following CDRs: HC-CDR1 having the amino acid sequence of SEQID NO:42 HC-CDR2 having the amino acid sequence of SEQ ID NO:45 HC-CDR3having the amino acid sequence of SEQ ID NO:48; and a VL region havingthe following CDRs: LC-CDR1 having the amino acid sequence of SEQ IDNO:88 LC-CDR2 having the amino acid sequence of SEQ ID NO:92 LC-CDR3having the amino acid sequence of SEQ ID NO:95; or (j) a VH regionhaving the following CDRs: HC-CDR1 having the amino acid sequence of SEQID NO:41 HC-CDR2 having the amino acid sequence of SEQ ID NO:44 HC-CDR3having the amino acid sequence of SEQ ID NO:47; and a VL region havingthe following CDRs: LC-CDR1 having the amino acid sequence of SEQ IDNO:88 LC-CDR2 having the amino acid sequence of SEQ ID NO:92 LC-CDR3having the amino acid sequence of SEQ ID NO:98.
 12. The method accordingto claim 10, wherein the antigen-binding molecule comprises: (a) a VHregion comprising an amino acid sequence having at least 70% sequenceidentity to the amino acid sequence of SEQ ID NO:24; and a VL regioncomprising an amino acid sequence having at least 70% sequence identityto the amino acid sequence of SEQ ID NO:74; or (b) a VH regioncomprising an amino acid sequence having at least 70% sequence identityto the amino acid sequence of SEQ ID NO:25; and a VL region comprisingan amino acid sequence having at least 70% sequence identity to theamino acid sequence of SEQ ID NO:75; or (c) a VH region comprising anamino acid sequence having at least 70% sequence identity to the aminoacid sequence of SEQ ID NO:26; and a VL region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:76; or (d) a VH region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:27; and a VL region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:77; or (e) a VH region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:28; and a VL region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:78; or (f) a VH region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:29; and a VL region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:78; or (g) a VH region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:30; and a VL region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:78; or (h) a VH region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:31; and a VL region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:79; or (i) a VH region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:32; and a VL region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:79; or (j) a VH region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:33; and a VL region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:80; or (k) a VH region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:34; and a VL region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:81; or (l) a VH region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:35; and a VL region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:82; or (m) a VH region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:36; and a VL region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:83; or (n) a VH region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:37; and a VL region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:84; or (o) a VH region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:38; and a VL region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:85; or (p) a VH region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:39; and a VL region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:86; or (q) a VH region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:40; and a VL region comprising an amino acidsequence having at least 70% sequence identity to the amino acidsequence of SEQ ID NO:87.
 13. The method according to claim 10, whereinthe cancer is selected from: a cancer comprising cells expressing anEGFR family member, a cancer comprising cells expressing HER3, a solidtumor, breast cancer, breast carcinoma, ductal carcinoma, gastriccancer, gastric carcinoma, gastric adenocarcinoma, colorectal cancer,colorectal carcinoma, colorectal adenocarcinoma, head and neck cancer,squamous cell carcinoma of the head and neck (SCCHN), lung cancer, lungadenocarcinoma, squamous cell lung carcinoma, ovarian cancer, ovariancarcinoma, ovarian serous adenocarcinoma, kidney cancer, renal cellcarcinoma, renal clear cell carcinoma, renal cell adenocarcinoma, renalpapillary cell carcinoma, pancreatic cancer, pancreatic adenocarcinoma,pancreatic ductal adenocarcinoma, cervical cancer, cervical squamouscell carcinoma, skin cancer, melanoma, esophageal cancer, esophagealadenocarcinoma, liver cancer, hepatocellular carcinoma,cholangiocarcinoma, uterine cancer, uterine corpus endometrialcarcinoma, thyroid cancer, thyroid carcinoma, pheochromocytoma,paraganglioma, bladder cancer, bladder urothelial carcinoma, prostatecancer, prostate adenocarcinoma, sarcoma and thymoma.